Microbial pathogenicity and virulence: definitions, parables and molecular aspects / A mikroorganizmusok patogenitása és virulenciája: definíciók, példázatok és molekuláris háttérismeretek.

Összefoglaló. A fertozo betegségek kóroki hátterének felderítésére irányuló törekvések hosszú idore tekintenek vissza. Fogalmakkal és követelményekkel (posztulátumokkal) igyekeztek körülírni, hogy egy mikroorganizmus mikor tekintheto egy adott fertozo betegség okozójának. Egy patogén rendszertani kategóriába tartozó mikroorganizmus kimutatása a betegbol önmagában még nem elegendo bizonyíték arra, hogy a betegségnek valóban az a kórokozója. Igazolni kell a továbbiakban, hogy rendelkezik azokkal a virulenciafaktoroknak nevezett tényezokkel, amelyek valójában képessé teszik az adott betegség kiváltására. Robert Koch idejében csak fenotípusos ismeretek álltak rendelkezésre, azok figyelembevételével fogalmazta meg posztulátumait. Késobb, a megszerzett molekuláris ismeretek birtokában, a posztulátumokat molekuláris szinten is értelmezték. A beteg személyét biológiai, szociális és pszichés egységként kezelo holisztikus megközelítésnek is eleget téve, a posztulátumokat a kórokozó mellett az esetben érintett gazdaszervezet egyedi tulajdonságainak figyelembevételével tovább szélesítették. A dolgozat a fenti kérdéseket példákkal illusztrálva tárgyalja, majd kitér a gyakorlati hasznosítás lehetoségeire. Orv Hetil. 2021; 162(50): 1991-1999. Summary. Efforts to explore the casual background of infectious diseases have been ambitioned for a long time. Terms and requirements (postulates) have been created to describe in which case a microorganism can be regarded as a causative agent of a given infectious disease. Demonstration of a representative of a pathogenic taxonomic category in the patient, however, does not prove its causative role in itself. It should also be verified if the microbe possesses the so-called virulence factors enabling it to trigger the given disease. At the time when Robert Koch formulated his postulates, only phenotypic characters were at his disposal. Later, in possession of a substantial genetic knowledge, the postulates have been adapted to molecular level. For having a holistic approach, the postulates have been extended also to the host's individual biological, social and psychological attributes. This paper discusses the above issues with examples for illustration, and outlines their practical applicabilities. Orv Hetil. 2021; 162(50): 1991-1999.

Virulence Traits of Inpatient Campylobacter jejuni Isolates, and a Transcriptomic Approach to Identify Potential Genes Maintaining Intracellular Survival.

There are still major gaps in our understanding of the bacterial factors that influence the outcomes of human Campylobacter jejuni infection. The aim of this study was to compare the virulence-associated features of 192 human C. jejuni strains isolated from hospitalized patients with diarrhoea (150/192, 78.1%), bloody diarrhoea (23/192, 11.9%), gastroenteritis (3/192, 1.6%), ulcerative colitis (3/192, 1.5%), and stomach ache (2/192, 1.0%). Traits were analysed with genotypic and phenotypic methods, including PCR and extracellular matrix protein (ECMP) binding, adhesion, and invasion capacities. Results were studied alongside patient symptoms, but no distinct links with them could be determined. Since the capacity of C. jejuni to invade host epithelial cells is one of its most enigmatic attributes, a high throughput transcriptomic analysis was performed in the third hour of internalization with a C. jejuni strain originally isolated from bloody diarrhoea. Characteristic groups of genes were significantly upregulated, outlining a survival strategy of internalized C. jejuni comprising genes related (1) to oxidative stress; (2) to a protective sheath formed by the capsule, LOS, N-, and O- glycosylation systems; (3) to dynamic metabolic activity supported by different translocases and the membrane-integrated component of the flagellar apparatus; and (4) to hitherto unknown genes.

Characterization of Asymptomatic Bacteriuria Escherichia coli Isolates in Search of Alternative Strains for Efficient Bacterial Interference against Uropathogens.

Asymptomatic bacterial colonization of the urinary bladder (asymptomatic bacteriuria, ABU) can prevent bladder colonization by uropathogens and thus symptomatic urinary tract infection (UTI). Deliberate bladder colonization with Escherichia coli ABU isolate 83972 has been shown to outcompete uropathogens and prevent symptomatic UTI by bacterial interference. Many ABU isolates evolved from uropathogenic ancestors and, although attenuated, may still be able to express virulence-associated factors. Our aim was to screen for efficient and safe candidate strains that could be used as alternatives to E. coli 83972 for preventive and therapeutic bladder colonization. To identify ABU E. coli strains with minimal virulence potential but maximal interference efficiency, we compared nine ABU isolates from diabetic patients regarding their virulence- and fitness-associated phenotypes in vitro, their virulence in a murine model of sepsis and their genome content. We identified strains in competitive growth experiments, which successfully interfere with colonization of ABU isolate 83972 or uropathogenic E. coli strain 536. Six isolates were able to outcompete E. coli 83972 and two of them also outcompeted UPEC 536 during growth in urine. Superior competitiveness was not simply a result of better growth abilities in urine, but seems also to involve expression of antagonistic factors. Competitiveness in urine did not correlate with the prevalence of determinants coding for adhesins, iron uptake, toxins, and antagonistic factors. Three ABU strains (isolates 61, 106, and 123) with superior competitiveness relative to ABU model strain 83972 display low in vivo virulence in a murine sepsis model, and susceptibility to antibiotics. They belong to different phylogroups and differ in the presence of ExPEC virulence- and fitness-associated genes. Importantly, they all lack marked cytotoxic activity and exhibit a high LD50 value in the sepsis model. These strains represent promising candidates for a more detailed assessment of relevant fitness traits in urine and their suitability for therapeutic bladder colonization.

Whole-Genome Draft Sequences of Nine Asymptomatic Escherichia coli Bacteriuria Isolates from Diabetic Patients.

Escherichia coli can colonize the urinary bladder without causing a disease response in the host. This asymptomatic bacteriuria (ABU) can protect against recurrent symptomatic urinary tract infection by virulent bacteria. Here, we report the whole-genome sequences of nine E. coli ABU isolates from diabetic patients.

Effect of primycin on growth-arrested cultures and cell integrity of Staphylococcus aureus.

Bactericidal effect against non-dividing bacteria is a very advantageous, but rare characteristic among antimicrobial agents, mostly possessed by those affecting the cell membrane. These kinds of agents can kill bacterial cells without lysis. We assessed these characteristics on primycin, a topical anti-staphylococcal agent highly effective against prevalent multiresistant strains, as it also acts on the cell membrane. In time-kill studies, primycin preserved its bactericidal activity against growth-arrested Staphylococcus aureus cultures. The bactericidal action was slower against growth-arrested cultures compared to the exponentially growing ones to different extents depending on the manner of arrest. The bactericidal effect was less influenced by stringent response and by protein synthesis inhibition, proving that it does not depend on metabolic activity. In contrast, uncoupling of the membrane potential predominantly slowed, and low temperature almost stopped killing of bacteria. In consideration of published data, these facts suggest that the antibacterial action of primycin involves disrupting of the membrane potential, and is predominantly influenced by the membrane fluidity. Optical density measurements and transmission electron microscopy verified that primycin kills bacterial cells without lysis. These results reveal favorable characteristics of primycin and point to, and broaden the knowledge on its membrane-targeted effect.

Antimicrobial and Virulence-Modulating Effects of Clove Essential Oil on the Foodborne Pathogen Campylobacter jejuni.

Our study investigated the antimicrobial action of clove (Syzygium aromaticum) essential oil (EO) on the zoonotic pathogen Campylobacter jejuni After confirming the clove essential oil's general antibacterial effect, we analyzed the reference strain Campylobacter jejuni NCTC 11168. Phenotypic, proteomic, and transcriptomic methods were used to reveal changes in cell morphology and functions when exposed to sublethal concentrations of clove EO. The normally curved cells showed markedly straightened and shrunken morphology on the scanning electron micrographs as a result of stress. Although, oxidative stress, as a generally accepted response to essential oils, was also present, the dominance of a general stress response was demonstrated by reverse transcription-PCR (RT-PCR). The results of RT-PCR and two-dimensional (2D) PAGE revealed that clove oil perturbs the expression of virulence-associated genes taking part in the synthesis of flagella, PEB1, PEB4, lipopolysaccharide (LPS), and serine protease. Loss of motility was also detected by a phenotypic test. Bioautographic analysis revealed that besides its major component, eugenol, at least four other spots of clove EO possessed bactericidal activity against C. jejuni Our findings show that clove EO has a marked antibacterial and potential virulence-modulating effect on C. jejuni IMPORTANCE: This study demonstrates that the components of clove essential oil influence not only the expression of general stress genes but also the expression of virulence-associated genes. Based on this finding, alternative strategies can be worked on to control this important foodborne pathogen.

A modified bioautographic method for antibacterial component screening against anaerobic and microaerophilic bacteria.

Direct bioautography is a useful method to identify antimicrobial compounds with potential therapeutic importance. Because of technical limitations till now, it has been applied only for aerobic bacteria. In this work we present the modification of the original method by which antimicrobial screening of bacteria requiring modified atmosphere became feasible by direct bioautography. Here we demonstrate its applicability by testing three anaerobic Clostridium perfringens and three microaerophilic Campylobacter jejuni strains against two essential oils, clove and thyme. Antimicrobial component profiles of clove and thyme essential oils against these two medically important pathogenic bacteria were compared and significant differences were revealed in their inhibition capacities. Linalool, a component of thyme essential oil exerted a more expressed antibacterial activity against C. perfringens than against C. jejuni. Our results demonstrate that direct bioautography is not only suitable for testing aerobic bacteria, but by applying the presently described modified version it can also contribute to the quest to find novel antimicrobial agents against multidrug resistant anaerobic and microaerophilic bacteria.

Identification and characterization of CTX-M-15 producing Klebsiella pneumoniae clone ST101 in a Hungarian university teaching hospital.

We investigated the molecular epidemiology of extended spectrum ß-lactamase (ESBL) producing Klebsiella pneumoniae isolates derived from the teaching hospitals of University of Pécs, Pécs, Hungary in the time period 2004-2008. Molecular typing, antimicrobial susceptibility testing, detection of common ß-lactamase genes (bla(CTX-M), bla(TEM) and bla(SHV)) and virulence associated traits (hypermucoviscosity, magA, k2a, rmpA, siderophores, type 1 and 3 fimbria, biofilm formation, serum resistance) were performed for 102 isolates. The results showed the presence of three major ciprofloxacin resistant CTX-M-15 producing clones (ST15 n = 69, ST101 n = 10, and ST147 n = 9), of which ST15 was predominant and universally widespread. Considering distribution in time and place, ST101 and ST147 were detected at fewer inpatient units and within a narrower time frame, as compared to ST15. Beside major clones, eleven minor clones were identified, and were shown to harbour the following ß-lactamase genes: six clones carried bla(CTX-M), four clones harboured bla(SHV-5) and one clone possessed both bla(CTX-M) and ESBL type bla(SHV). Among the SHV-5 producing K. pneumoniae clones a novel sequence type was found, namely ST1193, which harboured a unique infB allele. Different virulence factor content and peculiar antimicrobial susceptibility profile were characteristic for each clone. In contrast to major clone isolates, which showed high level resistance to ciprofloxacin, minor clone isolates displayed significantly lower MIC values for ciprofloxacin suggesting a role for fluoroquinolones in the dissemination of the major K. pneumoniae clones. This is the first description of the CTX-M-15 producing K. pneumoniae clone ST101 in Hungary.

Improved isolation protocol to detect high molecular weight polysaccharide structures of Campylobacter jejuni.

Simple detection of high molecular weight, LPS-like structures of Campylobacter jejuni is still an unsolved problem. A phenol-free extraction method for the detection of HMW polysaccharide was developed without the need for Western blot. This method provides a reliable technique for large-scale screening and comparative characterization study of different isolates.

Re-evaluation of in vitro activity of primycin against prevalent multiresistant bacteria.

With the increasing emergence of antibiotic resistances old antibiotics became a valuable source to find agents suitable to address this problem. More than 20 years after the last report, our purpose was to re-evaluate the in vitro antibacterial activity of the topical agent primycin against current important bacterial pathogens. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of primycin were tested in comparison with agents widely applied topically, and with those of mupirocin and vancomycin, the topical and the non-topical gold-standard anti-MRSA agents. Primycin was ineffective (MIC>64 µg/ml) against all the Gram-negative isolates tested. On the other hand, all the tested Gram-positive isolates were susceptible with MIC90 values of 0.06 µg/ml for staphylococci and 0.5-1 µg/ml for enterococci, streptococci, and P. acnes isolates, including all the multiresistant strains. Against MRSA isolates primycin showed slightly higher activity than mupirocin, and inhibited the mupirocin-resistant strains also. MBC90 values ranged from 0.25 to 2 µg/ml for the investigated Gram-positive species. The bactericidal effect proved to be concentration-dependent in time-kill experiments. Spontaneous resistant mutants did not emerge in single-step mutation experiments and the resistance development was very slow by serial passaging. Passaged S. aureus strains showing increased primycin MIC values exhibited elevated vancomycin and daptomycin MIC values also. Though elucidation of the mechanisms behind warrants further investigations, these correlations can be related to development of vancomycin-intermediate phenotype. From the point of view of medical practice it is noteworthy that the increased primycin MIC values remained far below the concentration accessible by local application of the agent. These data make primycin a remarkable object of further investigations as well as a promising candidate for topical application against multiresistant Gram-positive pathogens.

Cross-protection provided by live Shigella mutants lacking major antigens.

The immune response elicited by Shigella infections is dominated by serotype-specific antibodies recognizing the LPS O-antigens. Although a marked antibody response to invasion plasmid antigens (Ipa-s) shared by all virulent strains is also induced, the varying level of immunity elicited by natural infections is serotype-restricted. Previous vaccines have tried to mimic and achieve this serotype-specific, infection-induced immunity. As, however, the four Shigella species can express 50 different types of O-antigens, current approaches with the aim to induce a broad coverage use a mixture of the most common O-antigens combined in single vaccines. In the current study we present data on an alternative approach to generate immunity protective against multiple serotypes. Mutants lacking both major immune-determinant structures (i.e. the Ipa and O-antigens) were not only highly attenuated, but, unlike their avirulent counterparts still expressing these antigens, elicited a protective immune response to heterologous serotypes in a murine model. Evidence is provided that protection was mediated by the enhanced immunogenic potential of minor conserved antigens. Furthermore, the rough, non-invasive double mutants triggered an immune response different from that induced by the smooth, invasive strains regarding the isotype of antibodies generated. These non-invasive, rough mutants may represent promising candidates for further development into live vaccines for the prophylaxis of bacillary dysentery in areas with multiple endemic serotypes.

Mobilisation and remobilisation of a large archetypal pathogenicity island of uropathogenic Escherichia coli in vitro support the role of conjugation for horizontal transfer of genomic islands.

BACKGROUND: A substantial amount of data has been accumulated supporting the important role of genomic islands (GEIs)--including pathogenicity islands (PAIs)--in bacterial genome plasticity and the evolution of bacterial pathogens. Their instability and the high level sequence similarity of different (partial) islands suggest an exchange of PAIs between strains of the same or even different bacterial species by horizontal gene transfer (HGT). Transfer events of archetypal large genomic islands of enterobacteria which often lack genes required for mobilisation or transfer have been rarely investigated so far. RESULTS: To study mobilisation of such large genomic regions in prototypic uropathogenic E. coli (UPEC) strain 536, PAI II536 was supplemented with the mobRP4 region, an origin of replication (oriVR6K), an origin of transfer (oriTRP4) and a chloramphenicol resistance selection marker. In the presence of helper plasmid RP4, conjugative transfer of the 107-kb PAI II536 construct occured from strain 536 into an E. coli K-12 recipient. In transconjugants, PAI II536 existed either as a cytoplasmic circular intermediate (CI) or integrated site-specifically into the recipient's chromosome at the leuX tRNA gene. This locus is the chromosomal integration site of PAI II536 in UPEC strain 536. From the E. coli K-12 recipient, the chromosomal PAI II536 construct as well as the CIs could be successfully remobilised and inserted into leuX in a PAI II536 deletion mutant of E. coli 536. CONCLUSIONS: Our results corroborate that mobilisation and conjugal transfer may contribute to evolution of bacterial pathogens through horizontal transfer of large chromosomal regions such as PAIs. Stabilisation of these mobile genetic elements in the bacterial chromosome result from selective loss of mobilisation and transfer functions of genomic islands.

Differential effects and interactions of endogenous and horizontally acquired H-NS-like proteins in pathogenic Escherichia coli.

The nucleoid-associated protein H-NS is important for gene regulation in Escherichia coli. We have studied H-NS interaction with StpA and an uncharacterized H-NS-like protein, Hfp, in the uropathogenic E. coli isolate 536 that expresses all three nucleoid-associated proteins. We found distinct interactions of the three proteins at the protein level, resulting in the formation of heteromers, as well as differences in their gene expression at the transcriptional level. Mutants lacking either StpA or Hfp alone did not exhibit a phenotype at 37 degrees C, which is consistent with a low level of expression at that temperature. Expression of the hfp and stpA genes was found to be induced by apparently diametrical conditions, and StpA and Hfp levels could be correlated to modulatory effects on the expression of different H-NS targets, the bgl operon and operons for virulence factors such as fimbriae and capsular polysaccharide. The hns/hfp and hns/stpA double mutants displayed severe growth defects at low and high temperatures respectively. Our findings demonstrated different requirements for the alternative H-NS/Hfp/StpA combinations under these growth conditions. We propose that Hfp and StpA have distinct functions and roles in a dynamic pool of nucleoid-associated proteins that is adapting to requirements in a particular environment.

The SfaXII protein from newborn meningitis E. coli is involved in regulation of motility and type 1 fimbriae expression.

The genomes of pathogenic Escherichia coli may contain several different fimbrial operons. How bacteria regulate and coordinate the choice of fimbrial expression under different circumstances remains largely unanswered. In this report we have investigated the role of the sfaX(II) gene associated to the Sfa(II) fimbrial determinant in the E. coli isolate IHE3034. sfaX(II) belongs to a subfamily of genes, the 17k Da genes, located near different fimbrial operons in uropathogenic and newborn meningitis E. coli (NMEC) strains. Using the NMEC isolate IHE3034 and non-pathogenic E. coli strains we found that the sfaX(II) gene had an inhibitory effect on type 1 fimbriae expression. Down-regulation of type 1 fimbriae was exerted at transcriptional level both by inhibiting expression from the fimA promoter and by reducing the frequency of OFF-to-ON switching. The effect of sfaX(II) on expression of the recombinase FimB that catalyzes OFF-to-ON switching might explain the described reduction in percentage of ON cells. Moreover, expression of the sfaX(II) gene strongly influenced motility and flagella production of the NMEC isolate IHE3034. We propose that the sfaX(II) gene, and presumably other members in the 17 kDa gene family, may play a role in the control of virulence related gene expression in pathogenic E. coli.

Type 1 fimbriae, a colonization factor of uropathogenic Escherichia coli, are controlled by the metabolic sensor CRP-cAMP.

Type 1 fimbriae are a crucial factor for the virulence of uropathogenic Escherichia coli during the first steps of infection by mediating adhesion to epithelial cells. They are also required for the consequent colonization of the tissues and for invasion of the uroepithelium. Here, we studied the role of the specialized signal transduction system CRP-cAMP in the regulation of type 1 fimbriation. Although initially discovered by regulating carbohydrate metabolism, the CRP-cAMP complex controls a major regulatory network in Gram-negative bacteria, including a broad subset of genes spread into different functional categories of the cell. Our results indicate that CRP-cAMP plays a dual role in type 1 fimbriation, affecting both the phase variation process and fimA promoter activity, with an overall repressive outcome on fimbriation. The dissection of the regulatory pathway let us conclude that CRP-cAMP negatively affects FimB-mediated recombination by an indirect mechanism that requires DNA gyrase activity. Moreover, the underlying studies revealed that CRP-cAMP controls the expression of another global regulator in Gram-negative bacteria, the leucine-responsive protein Lrp. CRP-cAMP-mediated repression is limiting the switch from the non-fimbriated to the fimbriated state. Consistently, a drop in the intracellular concentration of cAMP due to altered physiological conditions (e.g. growth in presence of glucose) increases the percentage of fimbriated cells in the bacterial population. We also provide evidence that the repression of type 1 fimbriae by CRP-cAMP occurs during fast growth conditions (logarithmic phase) and is alleviated during slow growth (stationary phase), which is consistent with an involvement of type 1 fimbriae in the adaptation to stress conditions by promoting biofilm growth or entry into host cells. Our work suggests that the metabolic sensor CRP-cAMP plays a role in coupling the expression of type 1 fimbriae to environmental conditions, thereby also affecting subsequent attachment and colonization of host tissues.

"Gently rough": the vaccine potential of a Salmonella enterica regulatory lipopolysaccharide mutant.

BACKGROUND: An alternative to multivalent vaccines could be to construct strains capable of conferring broad protection through shared antigens. Down-regulation of immunodominant major antigens has been proposed to enhance the immunogenicity of conserved antigens. METHODS: The protection provided by an aroA as well as structural and regulatory lipopolysaccharide (LPS) mutants of Salmonella enterica serovar Typhimurium against homologous and heterologous challenges was assessed in the murine model of typhoid. The reactivity and cross-reactivity of the immune sera raised was tested by enzyme-linked immunospot assay and immunoblots. Conserved outer membrane proteins were identified by mass spectrometry. RESULTS: Unlike any structural LPS mutants, the regulatory mutant lacking RfaH was finely balanced between safety and immunogenicity, and its vaccine potential was comparable to that of the well-characterized DeltaaroA mutant. Loss of the transcriptional antiterminator RfaH resulted in a heterogeneous length of LPS chains, designated here as the "gently rough" phenotype. Our study also provides evidence that the rough phenotype enhances the immunogenicity of minor antigens, which may improve cross-protection against heterologous bacteria. A panel of conserved antigens shared by members of the Enterobacteriaceae family was identified as abundant porins and lipoprotein antigens. CONCLUSIONS: Fine-tuned down-regulation of immunodominant epitopes can create live vaccine strains that are not only desirably attenuated but that also exhibit an improved cross-protective potential.

Strategies for the development of vaccines conferring broad-spectrum protection.

Efficacious vaccination needs to confer protection against the vast majority of pathogens capable of causing a particular disease. Development of such vaccines is hindered by the great variability of microbes. Most pathogens have evolved variants that are able to express non-uniform surface structures. Naturally, evolutionary pressure has selected the most immunogenic antigens to be the most versatile. A combination of these multiform surface antigens forms the basis of classification of microbes into serotypes. Unfortunately, immune response in most cases is serotype-dependent, i.e. cross-protection among serotypes/serogroups of a given pathogen is limited. This review focuses on the strategies used for the engineering of broad-protective vaccine candidates, i.e., vaccines that induce a global, serotype-independent protection. The most plausible approach is to immunize with a multivalent vaccine containing different serotypes or purified serotype-determining antigens of a given pathogen. This arrangement is, however, efficient only against those microbes that have a limited number of serotypes, or few serotypes are responsible for the majority of the infections. Instead of using multivalent vaccine cocktails, cross-protective capacity of vaccine strains could be improved by making the conserved (i.e., shared by all variants) antigens more immunogenic. Elimination or down-regulation of the non-uniform antigens may increase immunogenicity of conserved minor antigens in vaccine candidates. Alternatively, shared antigens might be over-expressed in homologous or heterologous attenuated strains. Finally, purified conserved antigens could be used as subunit vaccines. In this paper, advantages and drawbacks of several such approaches will be reviewed.

Pathogenomics: an updated European Research Agenda.

The emerging genomic technologies and bioinformatics provide novel opportunities for studying life-threatening human pathogens and to develop new applications for the improvement of human and animal health and the prevention, treatment, and diagnosis of infections. Based on the ecology and population biology of pathogens and related organisms and their connection to epidemiology, more accurate typing technologies and approaches will lead to better means of disease control. The analysis of the genome plasticity and gene pools of pathogenic bacteria including antigenic diversity and antigenic variation results in more effective vaccines and vaccine implementation programs. The study of newly identified and uncultivated microorganisms enables the identification of new threats. The scrutiny of the metabolism of the pathogen in the host allows the identification of new targets for anti-infectives and therapeutic approaches. The development of modulators of host responses and mediators of host damage will be facilitated by the research on interactions of microbes and hosts, including mechanisms of host damage, acute and chronic relationships as well as commensalisms. The study of multiple pathogenic and non-pathogenic microbes interacting in the host will improve the management of multiple infections and will allow probiotic and prebiotic interventions. Needless to iterate, the application of the results of improved prevention and treatment of infections into clinical tests will have a positive impact on the management of human and animal disease. The Pathogenomics Research Agenda draws on discussions with experts of the Network of Excellence "EuroPathoGenomics" at the management board meeting of the project held during 18-21 April 2007, in the Villa Vigoni, Menaggio, Italy. Based on a proposed European Research Agenda in the field of pathogenomics by the ERA-NET PathoGenoMics the meeting's participants updated the established list of topics as the research agenda for the future.