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A 24-year-old patient from Cameroon presented to our hospital because of a foreign structure in her left eye. To our knowledge, for the first time, fluorescent microscopy revealed motile microfilariae, and the diagnosis of loiasis was established. Despite substantial microfilaremia, eosinophilia only unmasked after the initiation of antiparasitic therapy.
Acinetobacter baumannii virulence is mediated by the concerted action of three phospholipases D
(2015)
Acinetobacter baumannii causes a broad range of opportunistic infections in humans. Its success as an emerging pathogen is due to a combination of increasing antibiotic resistance, environmental persistence and adaptation to the human host. To date very little is known about the molecular basis of the latter. Here we demonstrate that A. baumannii can use phosphatidylcholine, an integral part of human cell membranes, as sole carbon and energy source. We report on the identification of three phospholipases belonging to the PLD superfamily. PLD1 and PLD2 appear restricted to the bacteria and display the general features of bacterial phospholipases D. They possess two PLDc_2 PFAM domains each encompassing the HxKx4Dx6GS/GGxN (HKD) motif necessary for forming the catalytic core. The third candidate, PLD3, is found in bacteria as well as in eukaryotes and harbours only one PLDc_2 PFAM domain and one conserved HKD motif, which however do not overlap. Employing a markerless mutagenesis system for A. baumannii ATCC 19606T, we generated a full set of PLD knock-out mutants. Galleria mellonella infection studies as well as invasion experiments using A549 human lung epithelial cells revealed that the three PLDs act in a concerted manner as virulence factors and are playing an important role in host cell invasion.
The antiviral drugs tecovirimat, brincidofovir, and cidofovir are considered for mpox (monkeypox) treatment despite a lack of clinical evidence. Moreover, their use is affected by toxic side-effects (brincidofovir, cidofovir), limited availability (tecovirimat), and potentially by resistance formation. Hence, additional, readily available drugs are needed. Here, therapeutic concentrations of nitroxoline, a hydroxyquinoline antibiotic with a favourable safety profile in humans, inhibited the replication of 12 mpox virus isolates from the current outbreak in primary cultures of human keratinocytes and fibroblasts and a skin explant model by interference with host cell signalling. Tecovirimat, but not nitroxoline, treatment resulted in rapid resistance development. Nitroxoline remained effective against the tecovirimat-resistant strain and increased the anti-mpox virus activity of tecovirimat and brincidofovir. Moreover, nitroxoline inhibited bacterial and viral pathogens that are often co-transmitted with mpox. In conclusion, nitroxoline is a repurposing candidate for the treatment of mpox due to both antiviral and antimicrobial activity.
Incidence of an intracellular multiplication niche amongst Acinetobacter baumannii clinical isolates
(2021)
The spread of antibiotic resistant Acinetobacter baumannii poses a significant threat to public health worldwide. This nosocomial bacterial pathogen can be associated with life-threatening infections, particularly in intensive care units. A. baumannii is mainly described as an extracellular pathogen with restricted survival within cells. This study shows that a subset of A. baumannii clinical isolates extensively multiply within non-phagocytic immortalized and primary cells, without the induction of apoptosis, and with bacterial clusters visible up to 48 hours after infection. This phenotype was observed for the A. baumannii C4 strain associated with high mortality in a hospital outbreak, and the A. baumannii ABC141 strain which wasn’t isolated from an infection site but was found to be hyperinvasive. Intracellular multiplication of these A. baumannii strains occurred within spacious single membrane-bound vacuoles, labeled with the lysosomal associate membrane protein (LAMP1). However, these compartments excluded lysotracker, an indicator of acidic pH, suggesting that A. baumannii can divert its trafficking away from the lysosomal degradative pathway. These compartments were also devoid of autophagy features. A high-content microscopy screen of 43 additional A. baumannii clinical strains highlighted various phenotypes: (1) the majority of strains remained extracellular, (2) a significant proportion was capable of invasion and limited persistence, and (3) two strains efficiently multiplied within LAMP1-positive vacuoles, one of which was also hyperinvasive. These data identify an intracellular niche for specific A. baumannii clinical strains that enables extensive multiplication in an environment protected from host immune responses and out of reach from many antibiotics.
Importance Multidrug resistant Acinetobacter baumannii strains are associated with significant morbidity and mortality in hospitals world-wide. Understanding their pathogenicity is critical for improving therapeutics. Although A. baumannii can steadily adhere to surfaces and host cells, most bacteria remain extracellular. Recent studies have shown that a small proportion of bacteria can invade cells but present limited survival. We have found that some A. baumannii clinical isolates can establish a specialized intracellular niche that sustains extensive intracellular multiplication for a prolonged time without induction of cell death. We propose that this intracellular compartment allows A. baumannii to escape the cell’s normal degradative pathway, protecting bacteria from host immune responses and potentially hindering antibiotic accessibility. This may contribute to A. baumannii persistence, relapsing infections and enhanced mortality in susceptible patients. A high-content microscopy-based screen confirmed this pathogenicity trait is present in other clinical isolates. There is an urgent need for new antibiotics or alternative antimicrobial approaches, particularly to combat carbapenem-resistant A. baumannii. The discovery of an intracellular niche for this pathogen as well as hyperinvasive isolates may help guide the development of antimicrobial therapies and diagnostics in the future.
The antiviral drugs tecovirimat, brincidofovir, and cidofovir are considered for mpox (monkeypox) treatment despite a lack of clinical evidence. Moreover, their use is affected by toxic side-effects (brincidofovir, cidofovir), limited availability (tecovirimat), and potentially by resistance formation. Hence, additional, readily available drugs are needed. Here, therapeutic concentrations of nitroxoline, a hydroxyquinoline antibiotic with a favourable safety profile in humans, inhibited the replication of 12 mpox virus isolates from the current outbreak in primary cultures of human keratinocytes and fibroblasts and a skin explant model by interference with host cell signalling. Tecovirimat, but not nitroxoline, treatment resulted in rapid resistance development. Nitroxoline remained effective against the tecovirimat-resistant strain and increased the anti-mpox virus activity of tecovirimat and brincidofovir. Moreover, nitroxoline inhibited bacterial and viral pathogens that are often co-transmitted with mpox. In conclusion, nitroxoline is a repurposing candidate for the treatment of mpox due to both antiviral and antimicrobial activity.
Sequence type 131 (ST131) is one of the predominant Escherichia coli lineages among extraintestinal pathogenic E. coli (ExPEC) that causes a variety of diseases in humans and animals and frequently shows multidrug resistance. Here, we report the first genome sequence of an ST131-ExPEC strain from poultry carrying the plasmid-encoded colistin resistance gene mcr-1.
The objective of this study was to characterize blaOXA-23 harbouring Acinetobacter indicus-like strains from cattle including genomic and phylogenetic analyses, antimicrobial susceptibility testing and evaluation of pathogenicity in vitro and in vivo. Nasal and rectal swabs (n = 45) from cattle in Germany were screened for carbapenem-non-susceptible Acinetobacter spp. Thereby, two carbapenem resistant Acinetobacter spp. from the nasal cavities of two calves could be isolated. MALDI-TOF mass spectrometry and 16S rDNA sequencing identified these isolates as A. indicus-like. A phylogenetic tree based on partial rpoB sequences indicated closest relation of the two bovine isolates to the A. indicus type strain A648T and human clinical A. indicus isolates, while whole genome comparison revealed considerable intraspecies diversity. High mimimum inhibitory concentrations were observed for carbapenems and other antibiotics including fluoroquinolones and gentamicin. Whole genome sequencing and PCR mapping revealed that both isolates harboured blaOXA-23 localized on the chromosome and surrounded by interrupted Tn2008 transposon structures. Since the pathogenic potential of A. indicus is unknown, pathogenicity was assessed employing the Galleria (G.) mellonella infection model and an in vitro cytotoxicity assay using A549 human lung epithelial cells. Pathogenicity in vivo (G. mellonella killing assay) and in vitro (cytotoxicity assay) of the two A. indicus-like isolates was lower compared to A. baumannii ATCC 17978 and similar to A. lwoffii ATCC 15309. The reduced pathogenicity of A. indicus compared to A. baumannii correlated with the absence of important virulence genes encoding like phospholipase C1+C2, acinetobactin outer membrane protein BauA, RND-type efflux system proteins AdeRS and AdeAB or the trimeric autotransporter adhesin Ata. The emergence of carbapenem-resistant A. indicus-like strains from cattle carrying blaOXA-23 on transposable elements and revealing genetic relatedness to isolates from human clinical sources requires further investigations regarding the pathogenic potential, genomic characteristics, zoonotic risk and putative additional sources of this new Acinetobacter species.
Incidence of an intracellular multiplication niche among Acinetobacter baumannii clinical isolates
(2022)
The spread of antibiotic-resistant Acinetobacter baumannii poses a significant threat to public health worldwide. This nosocomial bacterial pathogen can be associated with life-threatening infections, particularly in intensive care units. A. baumannii is mainly described as an extracellular pathogen with restricted survival within cells. This study shows that a subset of A. baumannii clinical isolates extensively multiply within nonphagocytic immortalized and primary cells without the induction of apoptosis and with bacterial clusters visible up to 48 h after infection. This phenotype was observed for the A. baumannii C4 strain associated with high mortality in a hospital outbreak and the A. baumannii ABC141 strain, which was isolated from the skin but was found to be hyperinvasive. Intracellular multiplication of these A. baumannii strains occurred within spacious single membrane-bound vacuoles, labeled with the lysosomal associate membrane protein (LAMP1). However, these compartments excluded lysotracker, an indicator of acidic pH, suggesting that A. baumannii can divert its trafficking away from the lysosomal degradative pathway. These compartments were also devoid of autophagy features. A high-content microscopy screen of 43 additional A. baumannii clinical isolates highlighted various phenotypes, and (i) the majority of isolates remained extracellular, (ii) a significant proportion was capable of invasion and limited persistence, and (iii) three more isolates efficiently multiplied within LAMP1-positive vacuoles, one of which was also hyperinvasive. These data identify an intracellular niche for specific A. baumannii clinical isolates that enables extensive multiplication in an environment protected from host immune responses and out of reach of many antibiotics.
IMPORTANCE Multidrug-resistant Acinetobacter baumannii isolates are associated with significant morbidity and mortality in hospitals worldwide. Understanding their pathogenicity is critical for improving therapeutic management. Although A. baumannii can steadily adhere to surfaces and host cells, most bacteria remain extracellular. Recent studies have shown that a small proportion of bacteria can invade cells but present limited survival. We have found that some A. baumannii clinical isolates can establish a specialized intracellular niche that sustains extensive intracellular multiplication for a prolonged time without induction of cell death. We propose that this intracellular compartment allows A. baumannii to escape the cell’s normal degradative pathway, protecting bacteria from host immune responses and potentially hindering antibiotic accessibility. This may contribute to A. baumannii persistence, relapsing infections, and enhanced mortality in susceptible patients. A high-content microscopy-based screen confirmed that this pathogenicity trait is present in other clinical A. baumannii isolates. There is an urgent need for new antibiotics or alternative antimicrobial approaches, particularly to combat carbapenem-resistant A. baumannii. The discovery of an intracellular niche for this pathogen, as well as hyperinvasive isolates, may help guide the development of antimicrobial therapies and diagnostics in the future.
Molecular surveillance of carbapenem-resistant gram-negative bacteria in liver transplant candidates
(2021)
Background: Carbapenem-resistant Gram-negative bacteria (CRGN) cause life-threatening infections due to limited antimicrobial treatment options. The occurrence of CRGN is often linked to hospitalization and antimicrobial treatment but remains incompletely understood. CRGN are common in patients with severe illness (e.g., liver transplantation patients). Using whole-genome sequencing (WGS), we aimed to elucidate the evolution of CRGN in this vulnerable cohort and to reconstruct potential transmission routes.
Methods: From 351 patients evaluated for liver transplantation, 18 CRGN isolates (from 17 patients) were analyzed. Using WGS and bioinformatic analysis, genotypes and phylogenetic relationships were explored. Potential epidemiological links were assessed by analysis of patient charts.
Results: Carbapenem-resistant (CR) Klebsiella pneumoniae (n=9) and CR Pseudomonas aeruginosa (n=7) were the predominating pathogens. In silico analysis revealed that 14/18 CRGN did not harbor carbapenemase-coding genes, whereas in 4/18 CRGN, carbapenemases (VIM-1, VIM-2, OXA-232, and OXA-72) were detected. Among all isolates, there was no evidence of plasmid transfer-mediated carbapenem resistance. A close phylogenetic relatedness was found for three K. pneumoniae isolates. Although no epidemiological context was comprehensible for the CRGN isolates, evidence was found that the isolates resulted of a transmission of a carbapenem-susceptible ancestor before individual radiation into CRGN.
Conclusion: The integrative epidemiological study reveals a high diversity of CRGN in liver cirrhosis patients. Mutation of carbapenem-susceptible ancestors appears to be the dominant way of CR acquisition rather than in-hospital transmission of CRGN or carbapenemase-encoding genetic elements. This study underlines the need to avoid transmission of carbapenem-susceptible ancestors in vulnerable patient cohorts.
OXA-48 is the most common carbapenemase in Enterobacterales in Germany and one of the most frequent carbapenemases worldwide. Several reports have associated blaOXA–48 with a virulent host phenotype. To challenge this hypothesis, 35 OXA-48-producing clinical isolates of Escherichia coli (n = 15) and Klebsiella pneumoniae (n = 20) were studied in vitro, in vivo employing the Galleria mellonella infection model and by whole-genome sequencing. Clinical isolates belonged to 7 different sequence types (STs) in E. coli and 12 different STs in K. pneumoniae. In 26/35 isolates blaOXA–48 was located on a 63 kb IncL plasmid. Horizontal gene transfer (HGT) to E. coli J53 was high in isolates with the 63 kb IncL plasmid (transconjugation frequency: ∼103/donor) but low in isolates with non-IncL plasmids (<10–6/donor). Several clinical isolates were both highly cytotoxic against human cells and virulent in vivo. However, 63 kb IncL transconjugants generated from these highly virulent isolates were not more cytotoxic or virulent when compared to the recipient strain. Additionally, no genes associated with virulence were detected by in silico analysis of OXA-48 plasmids. The 63 kb plasmid was highly stable and did not impair growth or fitness in E. coli J53. In conclusion, OXA-48 clinical isolates in Germany are diverse but typically harbor the same 63 kb IncL plasmid which has been reported worldwide. We demonstrate that this 63 kb IncL plasmid has a low fitness burden, high plasmid stability and can be transferred by highly efficient HGT which is likely the cause of the rapid dissemination of OXA-48 rather than the expansion of a single clone or gain of virulence.