Escherichia coli killing by epidemiologically successful sublineages of Shigella sonnei is mediated by colicins.

BACKGROUND: Shigella sp. are enteric pathogens which causes >125 million cases of shigellosis annually. S. sonnei accounts for about a quarter of those cases and is increasingly prevalent in industrialising nations. Being an enteric pathogen, S. sonnei benefits from outcompeting gut commensals such as Escherichia coli to establish itself and cause disease. There are numerous mechanisms that bacterial pathogens use to outcompete its rivals including molecules called colicins. A Type 6 Secretion System (T6SS) was recently described as contributing to E. coli killing in S. sonnei. METHODS: We used Bulk Phenotyping of Epidemiological Replicates (BPER) which combined bacterial Genome Wide Association Studies (bGWAS) and high throughput phenotyping on a collection of S. sonnei surveillance isolates to identify the genetic features associated with E. coli killing and explore their relationship with epidemiological behaviour. We further explored the presence of colicins and T6SS components in the isolates using genomics, laboratory experimentation, and proteomics. FINDINGS: Our bGWAS analysis returned known and novel colicin and colicin related genes as significantly associated with E. coli killing. In silico analyses identified key colicin clusters responsible for the killing phenotype associated with epidemiologically successful sub-lineages. The killing phenotype was not associated with the presence of a T6SS. Laboratory analyses confirmed the presence of the key colicin clusters and that killing was contact-independent. INTERPRETATION: Colicins are responsible for E. coli killing by S. sonnei, not a T6SS. This phenotype contributes to shaping the observed epidemiology of S. sonnei and may contribute to its increasing prevalence globally. BPER is an epidemiologically relevant approach to phenotypic testing that enables the rapid identification of genetic drivers of phenotypic changes, and assessment of their relevance to epidemiology in natural settings. FUNDING: Biotechnology and Biological Sciences Research Council, Biotechnology and Biological Sciences Research Council Doctoral Training Partnership studentship, Wellcome Trust, Medical Research Council (UK), French National Research Agency.

Shigella Serotypes Associated With Carriage in Humans Establish Persistent Infection in Zebrafish.

Shigella represents a paraphyletic group of enteroinvasive Escherichia coli. More than 40 Shigella serotypes have been reported. However, most cases within the men who have sex with men (MSM) community are attributed to 3 serotypes: Shigella sonnei unique serotype and Shigella flexneri 2a and 3a serotypes. Using the zebrafish model, we demonstrate that Shigella can establish persistent infection in vivo. Bacteria are not cleared by the immune system and become antibiotic tolerant. Establishment of persistent infection depends on the O-antigen, a key constituent of the bacterial surface and a serotype determinant. Representative isolates associated with MSM transmission persist in zebrafish, while representative isolates of a serotype not associated with MSM transmission do not. Isolates of a Shigella serotype establishing persistent infections elicited significantly less macrophage death in vivo than isolates of a serotype unable to persist. We conclude that zebrafish are a valuable platform to illuminate factors underlying establishment of Shigella persistent infection in humans.

The evolution and international spread of extensively drug resistant Shigella sonnei.

Shigella sonnei causes shigellosis, a severe gastrointestinal illness that is sexually transmissible among men who have sex with men (MSM). Multidrug resistance in S. sonnei is common including against World Health Organisation recommended treatment options, azithromycin, and ciprofloxacin. Recently, an MSM-associated outbreak of extended-spectrum ß-lactamase producing, extensively drug resistant S. sonnei was reported in the United Kingdom. Here, we aimed to identify the genetic basis, evolutionary history, and international dissemination of the outbreak strain. Our genomic epidemiological analyses of 3,304 isolates from the United Kingdom, Australia, Belgium, France, and the United States of America revealed an internationally connected outbreak with a most recent common ancestor in 2018 carrying a low-fitness cost resistance plasmid, previously observed in travel associated sublineages of S. flexneri. Our results highlight the persistent threat of horizontally transmitted antimicrobial resistance and the value of continuing to work towards early and open international sharing of genomic surveillance data.

The genomic epidemiology of Escherichia albertii infecting humans and birds in Great Britain.

Escherichia albertii is a recently identified gastrointestinal bacterial pathogen of humans and animals which is typically misidentified as pathotypes of diarrhoeagenic Escherichia coli or Shigella species and is generally only detected during genomic surveillance of other Enterobacteriaceae. The incidence of E. albertii is likely underestimated, and its epidemiology and clinical relevance are poorly characterised. Here, we whole genome sequenced E. albertii isolates from humans (n = 83) and birds (n = 79) isolated in Great Britain between 2000 and 2021 and analysed these alongside a broader public dataset (n = 475) to address these gaps. We found human and avian isolates typically (90%; 148/164) belonged to host-associated monophyletic groups with distinct virulence and antimicrobial resistance profiles. Overlaid patient epidemiological data suggested that human infection was likely related to travel and possibly foodborne transmission. The Shiga toxin encoding stx2f gene was associated with clinical disease (OR = 10.27, 95% CI = 2.98-35.45 p = 0.0002) in finches. Our results suggest that improved future surveillance will further elucidate disease ecology and public and animal health risks associated with E. albertii.

Accessory Genome Dynamics and Structural Variation of Shigella from Persistent Infections.

Shigellosis is a diarrheal disease caused mainly by Shigella flexneri and Shigella sonnei Infection is thought to be largely self-limiting, with short- to medium-term and serotype-specific immunity provided following clearance. However, cases of men who have sex with men (MSM)-associated shigellosis have been reported where Shigella of the same serotype were serially sampled from individuals between 1 and 1,862 days apart, possibly due to persistent carriage or reinfection with the same serotype. Here, we investigate the accessory genome dynamics of MSM-associated S. flexneri and S. sonnei isolates serially sampled from individual patients at various days apart to shed light on the adaptation of these important pathogens during infection. We find that pairs likely associated with persistent infection/carriage and with a smaller single nucleotide polymorphism (SNP) distance, demonstrated significantly less variation in accessory genome content than pairs likely associated with reinfection, and with a greater SNP distance. We observed antimicrobial resistance acquisition during Shigella carriage, including the gain of an extended-spectrum beta-lactamase gene during carriage. Finally, we explored large chromosomal structural variations and rearrangements in seven (five chronic and two reinfection associated) pairs of S. flexneri 3a isolates from an MSM-associated epidemic sublineage, which revealed variations at several common regions across isolate pairs, mediated by insertion sequence elements and comprising a distinct predicted functional profile. This study provides insight on the variation of accessory genome dynamics and large structural genomic changes in Shigella during persistent infection/carriage. In addition, we have also created a complete reference genome and biobanked isolate of the globally important pathogen, S. flexneri 3a.IMPORTANCEShigella spp. are Gram-negative bacteria that are the etiological agent of shigellosis, the second most common cause of diarrheal illness among children under the age of five in low-income countries. In high-income countries, shigellosis is also a sexually transmissible disease among men who have sex with men. Within the latter setting, we have captured prolonged and/or recurrent infection with shigellae of the same serotype, challenging the belief that Shigella infection is short lived and providing an early opportunity to study the evolution of the pathogen over the course of infection. Using this recently emerged transmission scenario, we comprehensively characterize the genomic changes that occur over the course of individual infection with Shigella and uncover a distinct functional profile of variable genomic regions, findings that have relevance for other Enterobacteriaceae.

Characterization of Extremely Drug-Resistant and Hypervirulent Acinetobacter baumannii AB030.

Acinetobacter baumannii is an important nosocomial bacterial pathogen. Multidrug-resistant isolates of A. baumannii are reported worldwide. Some A. baumannii isolates display resistance to nearly all antibiotics, making treatment of infections very challenging. As the need for new and effective antibiotics against A. baumannii becomes increasingly urgent, there is a need to understand the mechanisms of antibiotic resistance and virulence in this organism. In this work, comparative genomics was used to understand the mechanisms of antibiotic resistance and virulence in AB030, an extremely drug-resistant and hypervirulent strain of A. baumannii that is a representative of a recently emerged lineage of A. baumannii International Clone V. In order to characterize AB030, we carried out a genomic and phenotypic comparison with LAC-4, a previously described hyper-resistant and hypervirulent isolate. AB030 contains a number of antibiotic resistance- and virulence-associated genes that are not present in LAC-4. A number of these genes are present on mobile elements. This work shows the importance of characterizing the members of new lineages of A. baumannii in order to determine the development of antibiotic resistance and virulence in this organism.

A response regulator protein with antar domain, AvnR, in Acinetobacter baumannii ATCC 17978 impacts its virulence and amino acid metabolism.

Acinetobacter baumannii, a Gram-negative coccobacillus, is notorious for its involvement in opportunistic infections around the world. Its resistance to antibiotics makes treatment of infections challenging. In this study, we describe a novel response regulator protein, AvnR (A1S_2006) that regulates virulence-related traits in A. baumannii ATCC17978. Sequence analysis suggests that AvnR is a CheY-like response regulator and contains the RNA-binding ANTAR (AmiR and NasR transcription anti-termination regulators) domain. We show that AvnR plays a role in regulating biofilm formation (on glass and plastic surfaces), surface motility, adhesion to A549 cells as well as in nitrogen metabolism in A. baumannii. RNA-Seq analysis revealed that avnR deletion results in altered expression of more than 150 genes (116 upregulated and 42 downregulated). RNA-Seq data suggest that altered biofilm formation and surface motility observed in the avnR deletion mutant is likely mediated by previously unknown pathways. Of note, was the altered expression of genes predicted to be involved in amino acid transport and metabolism in avnR deletion mutant. Biolog phenotypic array showed that deletion of avnR hampered A. baumannii ATCC17978's ability to metabolize various nitrogen sources, particularly that of glutamic acid, serine, histidine, aspartic acid, isoleucine and arginine. Taken together our data show that AvnR, the first ANTAR protein described in A. baumannii, affects virulence phenotypes as well as its ability to metabolize nitrogen sources.

Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii.

The purpose of this study was to create single-copy gene expression systems for use in genomic manipulations of multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates of Acinetobacter baumannii In this study, mini-Tn7 vectors with zeocin and apramycin selection markers were created by cloning the ble and aac(3)-IV genes, respectively, enabling either inducible gene expression (pUC18T-mini-Tn7T-Zeo-LAC and pUC18T-mini-Tn7T-Apr-LAC) or expression from native or constitutive promoters (pUC18T-mini-Tn7T-Zeo and pUC18T-mini-Tn7T-Apr). The selection markers of these plasmids are contained within a Flp recombinase target (FRT) cassette, which can be used to obtain unmarked mini-Tn7 insertions upon introduction of a source of Flp recombinase. To this end, site-specific excision vectors pFLP2A and pFLP2Z (containing apramycin and zeocin selection markers, respectively) were created in this study as an accessory to the mini-Tn7 vectors described above. Combinations of these novel mini-Tn7 plasmids and their compatible pFLP2Z or pFLP2A accessory plasmid were used to generate unmarked insertions in MDR clinical isolates of A. baumannii In addition, several fluorescent markers were cloned and inserted into MDR and XDR clinical isolates of A. baumannii via these apramycin and zeocin mini-Tn7 constructs to demonstrate their application.IMPORTANCEAcinetobacter baumannii is a high-priority pathogen for which research on mechanisms of resistance and virulence is a critical need. Commonly used antibiotic selection markers are not suitable for use in MDR and XDR isolates of A. baumannii due to the high antibiotic resistance of these isolates, which poses a barrier to the study of this pathogen. This study demonstrates the practical potential of using apramycin and zeocin mini-Tn7- and Flp recombinase-encoded constructs to carry out genomic manipulations in clinical isolates of A. baumannii displaying MDR and XDR phenotypes.

The Anthelmintic Drug Niclosamide Synergizes with Colistin and Reverses Colistin Resistance in Gram-Negative Bacilli.

There is an urgent need for new therapies to overcome antimicrobial resistance especially in Gram-negative bacilli (GNB). Repurposing old U.S. Food and Drug Administration-approved drugs as complementary agents to existing antibiotics in a synergistic combination presents an attractive strategy. Here, we demonstrate that the anthelmintic drug niclosamide selectively synergized with the lipopeptide antibiotic colistin against colistin-susceptible but more importantly against colistin-resistant GNB, including clinical isolates that harbor the mcr-1 gene. Breakpoints for colistin susceptibility in resistant Gram-negative bacilli were reached in the presence of 1 µg/ml (3 µM) niclosamide. Reversal of colistin resistance was also observed in combinations of niclosamide and polymyxin B. Enhanced bacterial killing was evident for the combination, in comparison to colistin monotherapy, against resistant Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae Accumulating evidence in the literature, along with our results, strongly suggests the potential for the combination of niclosamide and colistin to treat colistin-resistant Gram-negative bacillary infections. Our finding is significant since colistin is an antibiotic of last resort for multidrug-resistant Gram-negative bacterial infections that are nonresponsive to conventional treatments. With the recent global dissemination of plasmid-encoded colistin resistance, the addition of niclosamide to colistin therapy may hold the key to overcome colistin resistance.

Signal Transduction Proteins in Acinetobacter baumannii: Role in Antibiotic Resistance, Virulence, and Potential as Drug Targets.

Acinetobacter baumannii is a notorious pathogen in health care settings around the world, primarily due to high resistance to antibiotics. A. baumannii also shows an impressive capability to adapt to harsh conditions in clinical settings, which contributes to its persistence in such conditions. Following their traditional role, the Two Component Systems (TCSs) present in A. baumannii play a crucial role in sensing and adapting to the changing environmental conditions. This provides A. baumannii with a greater chance of survival even in unfavorable conditions. Since all the TCSs characterized to date in A. baumannii play a role in its antibiotic resistance and virulence, understanding the underlying molecular mechanisms behind TCSs can help with a better understanding of the pathways that regulate these phenotypes. This can also guide efforts to target TCSs as novel drug targets. In this review, we discuss the roles of TCSs in A. baumannii, their molecular mechanisms, and most importantly, the potential of using small molecule inhibitors of TCSs as potential novel drug targets.

Tn7-Based Single-Copy Insertion Vectors for Acinetobacter baumannii.

Acinetobacter baumannii is considered a problematic Gram-negative pathogen due to its widespread resistance to antibiotics. Understanding of resistance mechanisms in A. baumannii is critical for designing new and effective therapeutic options. However, this is hampered by the lack of tools to carry out genetic manipulations in A. baumannii. Here, we describe methods to use a chromosomal mini-Tn7-based single-copy gene expression system in A. baumannii. This system can be effectively used for performing genetic complementation studies, for tagging with fluorescent proteins, or for reporter fusion assays.

KatG-Mediated Oxidation Leading to Reduced Susceptibility of Bacteria to Kanamycin.

Resistance to antibiotics has become a serious problem for society, and there are increasing efforts to understand the reasons for and sources of resistance. Bacterial-encoded enzymes and transport systems, both innate and acquired, are the most frequent culprits for the development of resistance, although in Mycobacterium tuberculosis, the catalase-peroxidase, KatG, has been linked to the activation of the antitubercular drug isoniazid. While investigating a possible link between aminoglycoside antibiotics and the induction of oxidative bursts, we observed that KatG reduces susceptibility to aminoglycosides. Investigation revealed that kanamycin served as an electron donor for the peroxidase reaction, reducing the oxidized ferryl intermediates of KatG to the resting state. Loss of electrons from kanamycin was accompanied by the addition of a single oxygen atom to the aminoglycoside. The oxidized form of kanamycin proved to be less effective as an antibiotic. Kanamycin inhibited the crystallization of KatG, but the smaller, structurally related glycoside maltose did cocrystallize with KatG, providing a suggestion as to the possible binding site of kanamycin.

Effect of Sodium Chloride on Surface-Associated Motility of Acinetobacter baumannii and the Role of AdeRS Two-Component System.

Acinetobacter baumannii is an important bacterial pathogen whose resistance to antibiotics is a growing concern worldwide. Among a wide array of resistance mechanisms displayed by A. baumannii, energy-dependent efflux of antibiotics by proteins belonging the resistance-nodulation-division family serves as an important one. AdeABC pump has been shown to be active in various clinical isolates. Regulation of this pump is controlled by the AdeRS two-component system. In this study, we show that the AdeRS system, in addition to modulating A. baumannii's antibiotic susceptibility, also plays a role in biofilm formation as well as surface-associated motility. We also show that AdeRS deletion mutant is more sensitive to saline stress. In particular, motility of A. baumannii ATCC17978 on agar surface is severely hampered at higher salt concentrations when AdeRS system is absent. Therefore, our study shows that AdeRS could be part of the A. baumannii adaptation strategy to salinity stress.

Effect of Incubation Temperature on Antibiotic Resistance and Virulence Factors of Acinetobacter baumannii ATCC 17978.

Acinetobacter baumannii is a notorious opportunistic pathogen that is prevalent mainly in hospital settings. The ability of A. baumannii to adapt and to survive in a range of environments has been a key factor for its persistence and success as an opportunistic pathogen. In this study, we investigated the effect of temperature on the clinically relevant phenotypes displayed by A. baumannii at 37°C and 28°C. Surface-associated motility was significantly reduced at 28°C, while biofilm formation on plastic surfaces was increased at 28°C. Decreased susceptibility to aztreonam and increased susceptibility to trimethoprim-sulfamethoxazole were observed at 28°C. No differences in virulence, as assayed in a Galleria mellonella model, were observed. Proteomic analysis showed differential expression of 629 proteins, of which 366 were upregulated and 263 were downregulated at 28°C. Upregulation of the Csu and iron uptake proteins at 28°C was a key finding for understanding some of the phenotypes displayed by A. baumannii at 28°C.

KatG and KatE confer Acinetobacter resistance to hydrogen peroxide but sensitize bacteria to killing by phagocytic respiratory burst.

AIMS: Catalase catalyzes the degradation of H2O2. Acinetobacter species have four predicted catalase genes, katA, katE, katG, and katX. The aims of the present study seek to determine which catalase(s) plays a predominant role in determining the resistance to H2O2, and to assess the role of catalase in Acinetobacter virulence. MAIN METHODS: Mutants of Acinetobacter baumannii and Acinetobacter nosocomialis with deficiencies in katA, katE, katG, and katX were tested for sensitivity to H2O2, either by halo assays or by liquid culture assays. Respiratory burst of neutrophils, in response to A. nosocomialis, was assessed by chemiluminescence to examine the effects of catalase on the production of reactive oxygen species (ROS) in neutrophils. Bacterial virulence was assessed using a Galleria mellonella larva infection model. KEY FINDINGS: The capacities of A. baumannii and A. nosocomialis to degrade H2O2 are largely dependent on katE. The resistance of both A. baumannii and A. nosocomialis to H2O2 is primarily determined by the katG gene, although katE also plays a minor role in H2O2 resistance. Bacteria lacking both the katG and katE genes exhibit the highest sensitivity to H2O2. While A. nosocomialis bacteria with katE and/or katG were able to decrease ROS production by neutrophils, these cells also induced a more robust respiratory burst in neutrophils than did cells deficient in both katE and katG. We also found that A. nosocomialis deficient in both katE and katG was more virulent than the wildtype A. nosocomialis strain. SIGNIFICANCE: Our findings suggest that inhibition of Acinetobacter catalase may help to overcome the resistance of Acinetobacter species to microbicidal H2O2 and facilitate bacterial disinfection.