Acinetobacter baumannii currently represents one of the most important nosocomial infection agent due to its multidrug-resistance and a propensity for the epidemic spread. The A. baumannii strains belonging to the international clonal lineages I (IC I) and II (IC II) are associated with the hospital outbreaks and a high virulence. However, the intra and inter lineage-specific features of strains belonging to these most worldwide spread A. baumannii clones are not thoroughly explored. In this study we have investigated a set of cell surface-related features of A. baumannii IC I (n = 20) and IC II (n = 16) lineage strains, representing 30 distinct pulsed-field gel electrophoresis types in the collection of clinical isolates obtained in Lithuanian tertiary care hospitals. We show that A. baumannii IC II strains are non-motile, do not form pellicle and display distinct capsular polysaccharide profile compared with the IC I strains. Moreover, in contrast to the overall highly hydrophobic IC I strains, IC II strains showed a greater variation in cell surface hydrophobicity. Within the IC II lineage, hydrophilic strains demonstrated reduced ability to form biofilm and adhere to the abiotic surfaces, also possessed twofold thicker cell wall and exhibited higher resistance to desiccation. Furthermore, these strains showed increased adherence to the lung epithelial cells and were more virulent in nematode and mouse infection model compared with the hydrophobic IC II strains. According to the polymerase chain reaction-based locus-typing, the reduction in hydrophobicity of IC II strains was not capsule or lipooligosaccharide locus type-dependent. Hence, this study shows that the most widespread A. baumannii clonal lineages I and II markedly differ in the series of cell surface-related phenotypes including the considerable phenotypic diversification of IC II strains at the intra-lineage level. These findings suggest that the genotypically related A. baumannii strains might evolve the features which could provide an advantage at the specific conditions outside or within the host.

Soil is one of the biggest reservoirs of microbial diversity, yet the processes that define the community dynamics are not fully understood. Apart from soil management being vital for agricultural purposes, it is also considered a favorable environment for the evolution and development of antimicrobial resistance, which is due to its high complexity and ongoing competition between the microorganisms. Different approaches to agricultural production might have specific outcomes for soil microbial community composition and antibiotic resistance phenotype. Therefore in this study we aimed to compare the soil microbiota and its resistome in conventional and organic farming systems that are continually influenced by the different treatment (inorganic fertilizers and pesticides vs. organic manure and no chemical pest management). The comparison of the soil microbial communities revealed no major differences among the main phyla of bacteria between the two farming styles with similar soil structure and pH. Only small differences between the lower taxa could be observed indicating that the soil community is stable, with minor shifts in composition being able to handle the different styles of treatment and fertilization. It is still unclear what level of intensity can change microbial composition but current conventional farming in Central Europe demonstrates acceptable level of intensity for soil bacterial communities. When the resistome of the soils was assessed by screening the total soil DNA for clinically relevant and soil-derived antibiotic resistance genes, a low variety of resistance determinants was detected (resistance to β-lactams, aminoglycosides, tetracycline, erythromycin, and rifampicin) with no clear preference for the soil farming type. The same soil samples were also used to isolate antibiotic resistant cultivable bacteria, which were predominated by highly resistant isolates of Pseudomonas, Stenotrophomonas, Sphingobacterium and Chryseobacterium genera. The resistance of these isolates was largely dependent on the efflux mechanisms, the soil Pseudomonas spp. relying mostly on RND, while Stenotrophomonas spp. and Chryseobacterium spp. on RND and ABC transporters.

BackgroundAcinetobacter baumannii is one of the most important opportunistic pathogens responsible for hospital acquired infections. It displays multi-drug resistance profile and has the ability to colonize surfaces and persist under harsh conditions. A. baumannii two-component signal transduction system BfmRS, consisting of response regulator BfmR and sensor kinase BfmS, has been implicated in the control of various virulence-related traits and has been suggested to act as a global modulator of A. baumannii physiology.ResultsHere, we assessed the role of BfmR regulator in pellicle formation and bacterial competition, features important for the establishment of A. baumannii in clinical environment. We show that BfmR is required for the pellicle formation of A. baumannii, as ΔbfmRS mutant lacked this phenotype. The loss of bfmRS also greatly reduced the secretion of A. baumannii Hcp protein, which is a component of T6SS secretion system. However, T6SS-mediated killing phenotype was not impaired in ΔbfmRS mutant. On the contrary, the same mutation resulted in the transcriptional activation of contact-dependent inhibition (CDI) system, which A. baumannii used to inhibit the growth of another clinical A. baumannii strain and a closely related species Acinetobacter baylyi.ConclusionsThe obtained results indicate that BfmR is not only required for the pellicle phenotype induction in A. baumannii, but also, due to the down-regulation of a CDI system, could allow the incorporation of other A. baumannii strains or related species, possibly increasing the likelihood of the pathogens’ survival.

BackgroundMultidrug resistant Acinetobacter baumannii is one of the major infection agents causing nosocomial pneumonia. Therefore, new therapeutic approaches against this bacterium are needed. Surface-exposed proteins from bacterial pathogens are implicated in a variety of virulence-related traits and are considered as promising candidates for vaccine development.ResultsWe show in this study that a large Blp1 protein from opportunistic pathogen A. baumannii is encoded in all examined clinical strains of globally spread international clonal lineages I (IC I) and II (IC II). The two blp1 gene variants exhibit lineage-specific distribution profile. By characterization of blp1 deletion mutants and their complementation with blp1 alleles we show that blp1 gene is required for A. baumannii biofilm formation and adhesion to epithelial cells in IC I strain but not in the IC II strain. Nevertheless both alleles are functional in restoring the deficient phenotypes of IC I strain. Moreover, the blp1 gene is required for the establishing of A. baumannii virulence phenotype in nematode and murine infection models. Additionally, we demonstrate that C-terminal 711 amino acid fragment of Blp1 elicits an efficient protection to lethal A. baumannii infection in a murine model using active and passive immunization approaches. Antiserum obtained against Blp1-specific antigen provides opsonophagocytic killing of A. baumannii in vitro.ConclusionsLineage-specific variants of surface-exposed components of bacterial pathogens complicate the development of new therapeutic approaches. Though we demonstrated different impact of Blp1 variants on adherence of IC I and IC II strains, Blp1-specific antiserum neutralized A. baumannii strains of both clonal lineages. Together with the observed increased survival rate in vaccinated mice these results indicate that A. baumannii Blp1 protein could be considered as a new vaccine candidate.

Multidrug resistant Acinetobacter baumannii shows a growing number of nosocomial infections worldwide during the last decade. The outer membrane vesicles (OMVs) produced by this bacterium draw increasing attention as a possible treatment target. OMVs have been implicated in the reduction of antibiotic level in the surrounding environment, transfer of virulence factors into the host cells, and induction of inflammatory response. Although the evidence on the involvement of OMVs in A. baumannii pathogenesis is currently growing, their role during inflammation is insufficiently explored. It is likely that bacteria, by secreting OMVs, can expand the area of their exposure and prepare surrounding matrix for infection. Here, we investigated the impact of A. baumannii OMVs on activation of macrophages in vitro. We show that OmpA protein present in A. baumannii OMVs substantially contributes to the proinflammatory response in J774 murine macrophages and to the cell death in both lung epithelium cells and macrophages. The loss of OmpA protein in OMVs, obtained from A. baumannii ∆ompA mutant, resulted in the altered expression of genes coding for IL-6, NLRP3 and IL-1β proinflammatory molecules in macrophages in vitro. These results imply that OmpA protein in bacterial OMVs could trigger a more intense proinflammatory response.

Acinetobacter baumannii is a nosocomial human pathogen of increasing concern due to its multidrug resistance profile. The outer membrane protein A (OmpA) is an abundant bacterial cell surface component involved in A. baumannii pathogenesis. It has been shown that the C-terminal domain of OmpA is located in the periplasm and non-covalently associates with the peptidoglycan layer via two conserved amino acids, thereby anchoring OmpA to the cell wall. Here, we investigated the role of one of the respective residues, D268 in OmpA of A. baumannii clinical strain Ab169, on its virulence characteristics by complementing the ΔompA mutant with the plasmid-borne ompAD268A allele. We show that while restoring the impaired biofilm formation of the ΔompA strain, the Ab169ompAD268A mutant tended to form bacterial filaments, indicating the abnormalities in cell division. Moreover, the Ab169 OmpA D268-mediated association to peptidoglycan was required for the manifestation of twitching motility, desiccation resistance, serum-induced killing, adhesion to epithelial cells and virulence in a nematode infection model, although it was dispensable for the uptake of β-lactam antibiotics by outer membrane vesicles. Overall, the results of this study demonstrate that the OmpA C-terminal domain-mediated association to peptidoglycan is critical for a number of virulent properties displayed by A. baumannii outside and within the host.

Aquatic environments are one of the most favorable settings for acquisition and dissemination of antimicrobial resistance. The aims of this study were twofold: (1) to isolate and identify antimicrobial resistant Gram-negative bacteria that are prevalent in fish kept in open aquaculture ponds and(2) to analyse their antimicrobial resistance profiles. One hundred and nineteen isolates from fish resistant to at least one antimicrobial were investigated. The most predominant genera were Pseudomonas, Chryseobacterium, Enterobacteriaceae, and Aeromonas. The highest numbers of multiresistant isolates were found within Pseudomonas spp., and the lowest, in Aeromonas spp.Resistance to beta-lactams was the most common among all of the bacterial genera. The absence or very low rates of resistance to fluoroquinolones, the small number of clinically important resistance genes, and bacterial taxonomic composition suggest that the most widespread resistant bacteria in aquacultured fish are autochthonous microbiota. Practical applicationsThe majority of antimicrobial resistant bacteria that are prevalent in aquacultured fish from open ponds can be treated as natural (autochthonous microbiota). In this study, only a minority of resistant bacteria detected in fish could be associated with polluted waters originating from animal farms or human healthcare facilities. Some strains, particularly the family Enterobacteriaceae that is found in fish, contain clinically important resistance determinants that potentially can be transmitted to human microbiota in cases of inappropriate processing of fish intended for food. With regard to antimicrobial resistant bacteria, measures for control of inflowing waters to aquaculture ponds should be taken in addition to monitoring fish microbiota and its' resistance should be investigated on a regular basis.

Neutrophilic granulocytes are the most abundant type of myeloid cells and form an essential part of the innate immune system. In vertebrates the first neutrophils are thought to originate during primitive hematopoiesis, which precedes hematopoietic stem cell formation. In zebrafish embryos, it has been suggested that primitive neutrophils may originate in two distinct sites, the anterior (ALPM) and posterior lateral plate mesoderm (PLPM). An ETS-family transcription factor Etsrp/Etv2/ER71 has been implicated in vasculogenesis and hematopoiesis in multiple vertebrates. However, its role during neutrophil development is not well understood. Here we demonstrate using zebrafish embryos that Etv2 has a specific cell-autonomous function during primitive neutropoiesis in the anterior lateral plate mesoderm (ALPM) but has little effect on erythropoiesis or the posterior lateral plate mesoderm (PLPM) expression of neutrophil marker myeloperoxidase mpo/mpx. Our results argue that ALPM-derived neutrophils originate from etv2-expressing cells which downregulate etv2 during neutropoiesis. We further show that Scl functions downstream of Etv2 in anterior neutropoiesis. Additionally, we demonstrate that mpx expression within the PLPM overlaps with gata1 expression, potentially marking the cells with a dual myelo-erythroid potential. Intriguingly, initiation of mpx expression in the PLPM is dependent on gata1 but not etv2 function. Our results demonstrate that mpx expression is controlled differently in the ALPM and PLPM regions and describe novel roles for etv2 and gata1 during primitive neutropoiesis.