Tracing clinically-relevant antimicrobial resistances in Acinetobacter baumannii-calcoaceticus complex across diverse environments: A study spanning clinical, livestock, and wastewater treatment settings.

Acinetobacter baumannii has become a prominent nosocomial pathogen, primarily owing to its remarkable ability to rapidly acquire resistance to a wide range of antimicrobial agents and its ability to persist in diverse environments. However, there is a lack of data on the molecular epidemiology and its potential implications for public health of A. baumannii strains exhibiting clinically significant resistances that originate from non-clinical environments. Therefore, the genetic characteristics and resistance mechanisms of 80 A. baumannii-calcoaceticus (ABC) complex isolates, sourced from environments associated with poultry and pig production, municipal wastewater treatment plants (WWTPs), and clinical settings, were investigated. In total, our study classified 54 isolates into 29 previously described sequence types (STs), while 26 isolates exhibited as-yet-unassigned STs. We identified a broad range of A. baumannii STs originating from poultry and pig production environments (e.g., ST10, ST238, ST240, ST267, ST345, ST370, ST372, ST1112 according to Pasteur scheme). These STs have also been documented in clinical settings worldwide, highlighting their clinical significance. These findings also raise concerns about the potential zoonotic transmission of certain STs associated with livestock environments. Furthermore, we observed that clinical isolates exhibited the highest diversity of antimicrobial resistance genes (ARGs). In contrast to non-clinical isolates, clinical isolates typically carried a significantly higher number of ARGs, ranging from 10 to 15. They were also the exclusive carriers of biocide resistance genes and acquired carbapenemases (blaOXA-23, blaOXA-58, blaOXA-72, blaGIM-1, blaNDM-1). Additionally, we observed that clinical strains displayed an increased capacity for carrying plasmids and undergoing genetic transformation. This heightened capability could be linked to the intense selective pressures commonly found within clinical settings. Our study provides comprehensive insights into essential aspects of ABC isolates originating from livestock-associated environments and clinical settings. We explored their resistance mechanisms and potential implications for public health, providing valuable knowledge for addressing these critical issues.

Genomic Evidence of mcr-1.26 IncX4 Plasmid Transmission between Poultry and Humans.

Colistin is still commonly used and misused in animal husbandry driving the evolution and dissemination of transmissible plasmid-mediated colistin resistance (mcr). mcr-1.26 is a rare variant and, so far, has only been detected in Escherichia coli obtained from a hospitalized patient in Germany in 2018. Recently, it was also notified in fecal samples from a pigeon in Lebanon. We report on the presence of 16 colistin-resistant, mcr-1.26-carrying extended-spectrum beta-lactamase (ESBL)-producing and commensal E. coli isolated from poultry samples in Germany, of which retail meat was the most common source. Short- and long-read genome sequencing and bioinformatic analyses revealed the location of mcr-1.26 exclusively on IncX4 plasmids. mcr-1.26 was identified on two different IncX4 plasmid types of 33 and 38 kb and was associated with an IS6-like element. Based on the genetic diversity of E. coli isolates, transmission of the mcr-1.26 resistance determinant is mediated by horizontal transfer of IncX4 plasmids, as confirmed by conjugation experiments. Notably, the 33-kb plasmid is highly similar to the plasmid reported for the human sample. Furthermore, we identified the acquisition of an additional beta-lactam resistance linked to a Tn2 transposon on the mcr-1.26 IncX4 plasmids of three isolates, indicating progressive plasmid evolution. Overall, all described mcr-1.26-carrying plasmids contain a highly conserved core genome necessary for colistin resistance development, transmission, replication, and maintenance. Variations in the plasmid sequences are mainly caused by the acquisition of insertion sequences and alteration in intergenic sequences or genes of unknown function. IMPORTANCE Evolutionary events causing the emergence of new resistances/variants are usually rare and challenging to predict. Conversely, common transmission events of widespread resistance determinants are quantifiable and predictable. One such example is the transmissible plasmid-mediated colistin resistance. The main determinant, mcr-1, has been notified in 2016 but has successfully established itself in multiple plasmid backbones in diverse bacterial species across all One Health sectors. So far, 34 variants of mcr-1 are described, of which some can be used for epidemiological tracing-back analysis to identify the origin and transmission dynamics of these genes. Here, we report the presence of the rare mcr-1.26 gene in E. coli isolated from poultry since 2014. Based on the temporal occurrence and high similarity of the plasmids between poultry and human isolates, our study provides first indications for poultry husbandry as the primary source of mcr-1.26 and its transmission between different niches.

Global colistin use: a review of the emergence of resistant Enterobacterales and the impact on their genetic basis.

The dramatic global rise of MDR and XDR Enterobacterales in human medicine forced clinicians to the reintroduction of colistin as last-resort drug. Meanwhile, colistin is used in the veterinary medicine since its discovery, leading to a steadily increasing prevalence of resistant isolates in the livestock and meat-based food sector. Consequently, transmission of resistant isolates from animals to humans, acquisition via food and exposure to colistin in the clinic are reasons for the increased prevalence of colistin-resistant Enterobacterales in humans in the last decades. Initially, resistance mechanisms were caused by mutations in chromosomal genes. However, since the discovery in 2015, the focus has shifted exclusively to mobile colistin resistances (mcr). This review will advance the understanding of chromosomal-mediated resistance mechanisms in Enterobacterales. We provide an overview about genes involved in colistin resistance and the current global situation of colistin-resistant Enterobacterales. A comparison of the global colistin use in veterinary and human medicine highlights the effort to reduce colistin sales in veterinary medicine under the One Health approach. In contrast, it uncovers the alarming rise in colistin consumption in human medicine due to the emergence of MDR Enterobacterales, which might be an important driver for the increasing emergence of chromosome-mediated colistin resistance.

Neuraminidase B controls neuraminidase A-dependent mucus production and evasion.

Binding of Streptococcus pneumoniae (Spn) to nasal mucus leads to entrapment and clearance via mucociliary activity during colonization. To identify Spn factors allowing for evasion of mucus binding, we used a solid-phase adherence assay with immobilized mucus of human and murine origin. Spn bound large mucus particles through interactions with carbohydrate moieties. Mutants lacking neuraminidase A (nanA) or neuraminidase B (nanB) showed increased mucus binding that correlated with diminished removal of terminal sialic acid residues on bound mucus. The non-additive activity of the two enzymes raised the question why Spn expresses two neuraminidases and suggested they function in the same pathway. Transcriptional analysis demonstrated expression of nanA depends on the enzymatic function of NanB. As transcription of nanA is increased in the presence of sialic acid, our findings suggest that sialic acid liberated from host glycoconjugates by the secreted enzyme NanB induces the expression of the cell-associated enzyme NanA. The absence of detectable mucus desialylation in the nanA mutant, in which NanB is still expressed, suggests that NanA is responsible for the bulk of the modification of host glycoconjugates. Thus, our studies describe a functional role for NanB in sialic acid sensing in the host. The contribution of the neuraminidases in vivo was then assessed in a murine model of colonization. Although mucus-binding mutants showed an early advantage, this was only observed in a competitive infection, suggesting a complex role of neuraminidases. Histologic examination of the upper respiratory tract demonstrated that Spn stimulates mucus production in a neuraminidase-dependent manner. Thus, an increase production of mucus containing secretions appears to be balanced, in vivo, by decreased mucus binding. We postulate that through the combined activity of its neuraminidases, Spn evades mucus binding and mucociliary clearance, which is needed to counter neuraminidase-mediated stimulation of mucus secretions.

Immune exclusion by naturally acquired secretory IgA against pneumococcal pilus-1.

Successful infection by mucosal pathogens requires overcoming the mucus barrier. To better understand this key step, we performed a survey of the interactions between human respiratory mucus and the human pathogen Streptococcus pneumoniae. Pneumococcal adherence to adult human nasal fluid was seen only by isolates expressing pilus-1. Robust binding was independent of pilus-1 adhesive properties but required Fab-dependent recognition of RrgB, the pilus shaft protein, by naturally acquired secretory IgA (sIgA). Pilus-1 binding by specific sIgA led to bacterial agglutination, but adherence required interaction of agglutinated pneumococci and entrapment in mucus particles. To test the effect of these interactions in vivo, pneumococci were preincubated with human sIgA before intranasal challenge in a mouse model of colonization. sIgA treatment resulted in rapid immune exclusion of pilus-expressing pneumococci. Our findings predict that immune exclusion would select for nonpiliated isolates in individuals who acquired RrgB-specific sIgA from prior episodes of colonization with piliated strains. Accordingly, genomic data comparing isolates carried by mothers and their children showed that mothers are less likely to be colonized with pilus-expressing strains. Our study provides a specific example of immune exclusion involving naturally acquired antibody in the human host, a major factor driving pneumococcal adaptation.

Contribution of Human Thrombospondin-1 to the Pathogenesis of Gram-Positive Bacteria.

A successful colonization of different compartments of the human host requires multifactorial contacts between bacterial surface proteins and host factors. Extracellular matrix proteins and matricellular proteins such as thrombospondin-1 play a pivotal role as adhesive substrates to ensure a strong interaction with pathobionts like the Gram-positive Streptococcus pneumoniae and Staphylococcus aureus. The human glycoprotein thrombospondin-1 is a component of the extracellular matrix and is highly abundant in the bloodstream during bacteremia. Human platelets secrete thrombospondin-1, which is then acquired by invading pathogens to facilitate colonization and immune evasion. Gram-positive bacteria express a broad spectrum of surface-exposed proteins, some of which also recognize thrombospondin-1. This review highlights the importance of thrombospondin-1 as an adhesion substrate to facilitate colonization, and we summarize the variety of thrombospondin-1-binding proteins of S. pneumoniae and S. aureus.

Interaction between the Staphylococcus aureus extracellular adherence protein Eap and its subdomains with platelets.

S. aureus associated bacteremia can lead to severe infections with high risk of mortality (e.g. sepsis, infective endocarditis). Many virulence factors and adhesins of S. aureus are known to directly interact with platelets. Extracellular adherence protein, Eap, one of the most important virulence factors in S. aureus mediated infections is a multi-tandem domain protein and has been shown to interact with almost all cell types in the human circulatory system. By using amine reactive fluorescent N-hydroxysuccinimidyl (NHS)-ester dyes and by direct detection with primary fluorescently conjugated anti-histidine (His-tag) antibodies against detect N-terminal His6, we show Eap subdomain Eap D3D4 specifically interacts and rapidly activates human platelets. Furthermore, we validate our finding by using site directed directional immobilization of Eap D3D4 through N-terminal His6 on nickel (II)-nitrilotriacetic acid (Ni-NTA) functionalized bacteriomimetic microbead arrays to visualize real-time platelet activation through calcium release assay. These methods offer an easily adoptable protocols for screening of S.aureus derived virulence factors and adhesins with platelets.

Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation.

Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection.

Serotype 3 pneumococci sequester platelet-derived human thrombospondin-1 via the adhesin and immune evasion protein Hic.

Streptococcus pneumoniae serotype 3 strains emerge frequently within clinical isolates of invasive diseases. Bacterial invasion into deeper tissues is associated with colonization and immune evasion mechanisms. Thus, pneumococci express a versatile repertoire of surface proteins sequestering and interacting specifically with components of the human extracellular matrix and serum. Hic, a PspC-like pneumococcal surface protein, possesses vitronectin and factor H binding activity. Here, we show that heterologously expressed Hic domains interact, similar to the classical PspC molecule, with human matricellular thrombospondin-1 (hTSP-1). Binding studies with isolated human thrombospondin-1 and various Hic domains suggest that the interaction between hTSP-1 and Hic differs from binding to vitronectin and factor H. Binding of Hic to hTSP-1 is inhibited by heparin and chondroitin sulfate A, indicating binding to the N-terminal globular domain or type I repeats of hTSP-1. Competitive inhibition experiments with other pneumococcal hTSP-1 adhesins demonstrated that PspC and PspC-like Hic recognize similar domains, whereas PavB and Hic can bind simultaneously to hTSP-1. In conclusion, Hic binds specifically hTSP-1; however, truncation in the N-terminal part of Hic decreases the binding activity, suggesting that the full length of the α-helical regions of Hic is required for an optimal interaction.

Pneumococcal Adhesins PavB and PspC Are Important for the Interplay with Human Thrombospondin-1.

The human matricellular glycoprotein thrombospondin-1 (hTSP-1) is released by activated platelets and mediates adhesion of Gram-positive bacteria to various host cells. In staphylococci, the adhesins extracellular adherence protein (Eap) and autolysin (Atl), both surface-exposed proteins containing repeating structures, were shown to be involved in the acquisition of hTSP-1 to the bacterial surface. The interaction partner(s) on the pneumococcal surface was hitherto unknown. Here, we demonstrate for the first time that pneumococcal adherence and virulence factor B (PavB) and pneumococcal surface protein C (PspC) are key players for the interaction of Streptococcus pneumoniae with matricellular hTSP-1. PavB and PspC are pneumococcal surface-exposed adhesins and virulence factors exhibiting repetitive sequences in their core structure. Heterologously expressed fragments of PavB and PspC containing repetitive structures exhibit hTSP-1 binding activity as shown by ELISA and surface plasmon resonance studies. Binding of hTSP-1 is charge-dependent and inhibited by heparin. Importantly, the deficiency in PavB and PspC reduces the recruitment of soluble hTSP-1 by pneumococci and decreases hTSP-1-mediated pneumococcal adherence to human epithelial cells. Platelet activation assays suggested that PavB and PspC are not involved in the activation of purified human platelets by pneumococci. In conclusion, this study indicates a pivotal role of PavB and PspC for pneumococcal recruitment of soluble hTSP-1 to the bacterial surface and binding of pneumococci to host cell-bound hTSP-1 during adhesion.

Repeating structures of the major staphylococcal autolysin are essential for the interaction with human thrombospondin 1 and vitronectin.

Human thrombospondin 1 (hTSP-1) is a matricellular glycoprotein facilitating bacterial adherence to and invasion into eukaryotic cells. However, the bacterial adhesin(s) remain elusive. In this study, we show a dose-dependent binding of soluble hTSP-1 to Gram-positive but not Gram-negative bacteria. Diminished binding of soluble hTSP-1 to proteolytically pretreated staphylococci suggested a proteinaceous nature of potential bacterial adhesin(s) for hTSP-1. A combination of separation of staphylococcal surface proteins by two-dimensional gel electrophoresis with a ligand overlay assay with hTSP-1 and identification of the target protein by mass spectrometry revealed the major staphylococcal autolysin Atl as a bacterial binding protein for hTSP-1. Binding experiments with heterologously expressed repeats of the AtlE amidase from Staphylococcus epidermidis suggest that the repeating sequences (R1ab-R2ab) of the N-acetyl-muramoyl-L-alanine amidase of Atl are essential for binding of hTSP-1. Atl has also been identified previously as a staphylococcal vitronectin (Vn)-binding protein. Similar to the interaction with hTSP-1, the R1ab-R2ab repeats of Atl are shown here to be crucial for the interaction of Atl with the complement inhibition and matrix protein Vn. Competition assays with hTSP-1 and Vn revealed the R1ab-R2ab repeats of AtlE as the common binding domain for both host proteins. Furthermore, Vn competes with hTSP-1 for binding to Atl repeats and vice versa. In conclusion, this study identifies the Atl repeats as bacterial adhesive structures interacting with the human glycoproteins hTSP-1 and Vn. Finally, this study provides insight into the molecular interplay between hTSP-1 and Vn, respectively, and a bacterial autolysin.