Metabolic Microenvironments Drive Microbial Differentiation and Antibiotic Resistance.

For bacteria, the transition from unicellular entities to multicellular biofilm communities generates distinct metabolic microenvironments. Dynamic and programmed metabolic responses allow the biofilms to react to local changes in nutrient levels. Moreover, metabolic adaptations contribute to phenotypic antibiotic resistance of the community, suggesting novel therapeutic approaches to target biofilms.

Use of Fourier-transform infrared spectroscopy for real-time outbreak investigation of OXA-48-producing Escherichia coli.

BACKGROUND: Efficient infection control during carbapenem-resistant Enterobacterales outbreaks demands rapid and simple techniques for outbreak investigations. WGS, the current gold standard for outbreak identification, is expensive, time-consuming and requires a high level of expertise. Fourier-transform infrared (FTIR) spectroscopy (IR Biotyper) is a rapid typing method based on infrared radiation applied to samples, which provides a highly specific absorption spectrum. OBJECTIVES: To investigate an outbreak of OXA-48-producing Escherichia coli in real-time using FTIR and subsequently compare the results with WGS. METHODS: Twenty-one isolates were collected during a nosocomial outbreak, and identification and antibiotic susceptibilities were confirmed by VITEK®2. FTIR was conducted for all isolates, and nine representative isolates were sequenced. RESULTS: FTIR was able to correctly determine the clonal relatedness of the isolates and to identify the outbreak cluster, as confirmed by WGS. By WGS, isolates in the main FTIR cluster belonged to the same MLST type and core-genome MLST type, and they harboured similar plasmids and resistance genes, whereas the singletons external to the FTIR cluster had different genetic content. CONCLUSIONS: FTIR can operate as a rapid, efficient and reliable first-line tool for outbreak investigations during a real-time ongoing E. coli outbreak, which can contribute to limiting the spread of pathogens.

Plasmid-encoded VCC-1 ß-lactamase in a clinical isolate of Aeromonas caviae.

Vibrio cholerae carbapenemase (VCC-1) is a chromosomal encoded class A carbapenemase thus far reported in environmental Vibrio cholerae isolates. Here, we report the first isolation of a blaVCC-1 -carrying Aeromonas caviae from a clinical sample in Israel. The isolate was resistant to all ß-lactam agents, including carbapenems. The blaVCC-1 was located on a large plasmid. GC content suggests that the origin of the blaVCC-1 gene is neither Aeromonas nor Vibrio spp. but an unknown progenitor.

Large-scale WGS of carbapenem-resistant Acinetobacter baumannii isolates reveals patterns of dissemination of ST clades associated with antibiotic resistance.

OBJECTIVES: To describe the population genetics and antibiotic resistance gene distribution of carbapenem-resistant Acinetobacter baumannii (CRAB) isolates causing infections in three Mediterranean countries. METHODS: Isolates were collected during the 2013-17 AIDA clinical trial in six hospitals in Israel, Greece and Italy. WGS, bioinformatic characterization and antibiotic resistance profiling were performed. RESULTS: In the 247 CRAB isolates characterized in this study, ST distribution varied by country: 29/31 (93.5%) Greek isolates, 34/41 (82.9%) Italian isolates and 70/175 (40.0%) Israeli isolates belonged to ST2. The identified ST2 isolates included eight distinct clades: 2C, 2D and 2H were significantly more common in Italy, while 2F was unique to Greece. The uncommon ST3 was not present among Greek isolates and constituted only 5/41 (12%) Italian isolates. On the other hand, it was much more common among Israeli isolates: 78/175 (44.6%) belonged to ST3. The vast majority of isolates, 240/247 (97.2%), were found to harbour acquired carbapenemases, primarily blaOXA-23. The chromosomal oxaAb (blaOXA-51-like) and ampC genes characteristic of this organism were also ubiquitous. Most (96.4%) ST3 isolates carried a broad-host-range plasmid IncP1α. CONCLUSIONS: The geographical differences in CRAB populations support the theory that clonal spread of CRAB leads to endemicity in hospitals and regions. The close association between antibiotic resistance genes and clades, and between plasmids and STs, suggest that de novo creation of MDR A. baumannii is rare. The clustering of antibiotic resistance genes and plasmids that is unique to each clade/ST, and nearly uniform within clades/STs, suggests that horizontal transmission is rare but crucial to the clade's/ST's success.

Indole Derivatives Maintain the Status Quo Between Beneficial Biofilms and Their Plant Hosts.

Biofilms formed by bacteria on plant roots play an important role in maintaining an optimal rhizosphere environment that supports plant growth and fitness. Bacillus subtilis is a potent plant growth promoter, forming biofilms that play a key role in protecting the host from fungal and bacterial infections. In this work, we demonstrate that the development of B. subtilis biofilms is antagonized by specific indole derivatives that accumulate during symbiotic interactions with plant hosts. Indole derivatives are more potent signals when the plant polysaccharide xylan serves as a carbon source, a mechanism to sustain beneficial biofilms at a biomass that can be supported by the plant. Moreover, B. subtilis biofilms formed by mutants resistant to indole derivatives become deleterious to the plants due to their capacity to consume and recycle plant polysaccharides. These results demonstrate how a dynamic metabolite-based dialogue can promote homeostasis between plant hosts and their beneficial biofilm communities.

An active ß-lactamase is a part of an orchestrated cell wall stress resistance network of Bacillus subtilis and related rhizosphere species.

A hallmark of the Gram-positive bacteria, such as the soil-dwelling bacterium Bacillus subtilis, is their cell wall. Here, we report that d-leucine and flavomycin, biofilm inhibitors targeting the cell wall, activate the ß-lactamase PenP. This ß-lactamase contributes to ampicillin resistance in B. subtilis under all conditions tested. In contrast, both Spo0A, a master regulator of nutritional stress, and the general cell wall stress response, differentially contribute to ß-lactam resistance under different conditions. To test whether ß-lactam resistance and ß-lactamase genes are widespread in other Bacilli, we isolated Bacillus species from undisturbed soils, and found that their genomes can encode up to five ß-lactamases with differentiated activity spectra. Surprisingly, the activity of environmental ß-lactamases and PenP, as well as the general stress response, resulted in a similarly reduced lag phase of the culture in the presence of ß-lactam antibiotics, with little or no impact on the logarithmic growth rate. The length of the lag phase may determine the outcome of the competition between ß-lactams and ß-lactamases producers. Overall, our work suggests that antibiotic resistance genes in B. subtilis and related species are ancient and widespread, and could be selected by interspecies competition in undisturbed soils.

The Plant Host Induces Antibiotic Production To Select the Most-Beneficial Colonizers.

Microbial ecosystems tightly associated with a eukaryotic host are widespread in nature. The genetic and metabolic networks of the eukaryotic hosts and the associated microbes have coevolved to form a symbiotic relationship. Both the Gram-positive Bacillus subtilis and the Gram-negative Serratia plymuthica can form biofilms on plant roots and thus can serve as a model system for the study of interspecies interactions in a host-associated ecosystem. We found that B. subtilis biofilms expand collectively and asymmetrically toward S. plymuthica, while expressing a nonribosomal antibiotic bacillaene and an extracellular protease. As a result, B. subtilis biofilms outcompeted S. plymuthica for successful colonization of the host. Strikingly, the plant host was able to enhance the efficiency of this killing by inducing bacillaene synthesis. In turn, B. subtilis biofilms increased the resistance of the plant host to pathogens. These results provide an example of how plant-bacterium symbiosis promotes the immune response of the plant host and the fitness of the associated bacteria.IMPORTANCE Our study sheds mechanistic light on how multicellular biofilm units compete to successfully colonize a eukaryote host, using B. subtilis microbial communities as our lens. The microbiota and its interactions with its host play various roles in the development and prevention of diseases. Using competing beneficial biofilms that are essential microbiota members on the plant host, we found that B. subtilis biofilms activate collective migration to capture their prey, followed by nonribosomal antibiotic synthesis. Plant hosts increase the efficiency of antibiotic production by B. subtilis biofilms, as they activate the synthesis of polyketides; therefore, our study provides evidence of a mechanism by which the host can indirectly select for beneficial microbiota members.

Modeling kinetic rate variation in third generation DNA sequencing data to detect putative modifications to DNA bases.

Current generation DNA sequencing instruments are moving closer to seamlessly sequencing genomes of entire populations as a routine part of scientific investigation. However, while significant inroads have been made identifying small nucleotide variation and structural variations in DNA that impact phenotypes of interest, progress has not been as dramatic regarding epigenetic changes and base-level damage to DNA, largely due to technological limitations in assaying all known and unknown types of modifications at genome scale. Recently, single-molecule real time (SMRT) sequencing has been reported to identify kinetic variation (KV) events that have been demonstrated to reflect epigenetic changes of every known type, providing a path forward for detecting base modifications as a routine part of sequencing. However, to date no statistical framework has been proposed to enhance the power to detect these events while also controlling for false-positive events. By modeling enzyme kinetics in the neighborhood of an arbitrary location in a genomic region of interest as a conditional random field, we provide a statistical framework for incorporating kinetic information at a test position of interest as well as at neighboring sites that help enhance the power to detect KV events. The performance of this and related models is explored, with the best-performing model applied to plasmid DNA isolated from Escherichia coli and mitochondrial DNA isolated from human brain tissue. We highlight widespread kinetic variation events, some of which strongly associate with known modification events, while others represent putative chemically modified sites of unknown types.

Zophobas morio larvae as a novel model for the study of Acinetobacter virulence and antimicrobial resistance.

The use of mammalian models for in vivo testing of bacterial virulence raises ethical concerns and is expensive and time-consuming. As an alternative, non-mammalian models are sought. Galleria mellonella larvae have been used as a model to study several bacterial pathogens. However, their maintenance is challenging, and commercial supply is low. In this study, we aimed to establish the Zophobas morio larvae as an alternative non-mammalian model for the evaluation of the pathogenicity and antimicrobial susceptibility of Acinetobacter baumannii. We infected Z. morio with Acinetobacter strains and determined the optimal temperature and inoculum. To visualize the bacterial distribution within the larvae, hematoxylin and eosin (H&E) staining was performed. Next, a survival model of infected larvae was established, and virulence was compared between strains. The effect of antimicrobial treatment in relation to antibiotic susceptibility was studied. Our results demonstrate that Z. morio can be used as a model system for in vivo studies of A. baumannii.

Contribution of active surveillance cultures to the control of hospital-acquired carbapenem-resistant Acinetobacter baumannii in an endemic hospital setting.

BACKGROUND: Despite the increasing rates of carbapenem-resistant Acinetobacter baumannii (CRAB) carriage among hospitalized patients in endemic settings, the role of active surveillance cultures and cohorting is still debated. We sought to determine the long-term effect of a multifaceted infection-control intervention on the incidence of CRAB in an endemic setting. METHODS: A prospective, quasi-experimental study was performed at a 670-bed, acute-care hospital. The study consisted of 4 phases. In phase I, basic infection control measures were used. In phase II, CRAB carriers were cohorted in a single ward with dedicated nursing and enhanced environmental cleaning. In phase III large-scale screening in high-risk units was implemented. Phase IV comprised a 15-month follow-up period. RESULTS: During the baseline period, the mean incidence rate (IDR) of CRAB was 44 per 100,000 patient days (95% CI, 37.7-54.1). No significant decrease was observed during phase II (IDR, 40.8 per 100,000 patient days; 95% CI, 30.0-56.7; P = .97). During phase III, despite high compliance with control measures, ongoing transmission in several wards was observed and the mean IDR was 53.9 per 100,000 patient days (95% CI, 40.5-72.2; P = .55). In phase IV, following the implementation of large-scale screening, a significant decrease in the mean IDR was observed (25.8 per 100,000 patient days; 95% CI, 19.9-33.5; P = .03). An overall reduction of CRAB rate was observed between phase I and phase IV (rate ratio, 0.6; 95% CI, 0.4-0.9; P < .001). CONCLUSIONS: The comprehensive intervention that included intensified control measures with routine active screening cultures was effective in reducing the incidence of CRAB in an endemic hospital setting.

Carbapenem-resistant Acinetobacter baumannii carrier detection: a simple and efficient protocol.

Timely detection of carbapenem-resistant Acinetobacter baumannii (CRAB) carriers is essential to direct infection control measures. In this work, we aimed to develop a practical protocol to detect CRAB from screening samples. To choose a selective medium that detects CRAB with high sensitivity and specificity, 111 A. baumannii clinical isolates were inoculated on three types of agar: mSuperCARBA (SC), CHROMagar Acinetobacter (CaA), and modified CHROMagar Acinetobacter (mCaA) containing 4.5 mg/mL meropenem. SC was non-selective, CaA was the most sensitive (100%), but only moderately specific (72%), and mCaA was highly specific (97%) and sensitive (98%). Confirmation of the carbapenem-resistant phenotype using PCR-based detection of blaOXA-23, blaOXA-24, and blaOXA-58 genes was specific but not sensitive, detecting only 58% of CRAB isolates. Identification of A. baumannii using either gyrB or blaOXA-51 PCR was excellent. Next, we used the same methodology in routine screening for CRAB carriage. mCaA had the best yield, with high sensitivity but moderate specificity to differentiate between CRAB and other carbapenem-resistant organisms. Skin sampling using sponges and 6 hour enrichment was highly sensitive (98%), while other body sites had poor sensitivity (27%- 41%). Shorter incubation had slightly lower yield, and longer incubation did not improve the detection. Performing PCR for blaOXA-51 and gyrB on colonies growing on modified mCaA differentiated between CRAB and other species with high accuracy (98% and 99%, respectively). Based on our results, we present a procedure for easy and reliable detection of CRAB carriage using skin sampling, short enrichment, selection on mCaA, and PCR-based identification. IMPORTANCE: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a substantial cause of nosocomial infections, classified among the most significant multidrug-resistant pathogens by the World Health Organization and by the US Centers for Disease Control. Limiting the spread of CRAB is an important goal of infection control, but laboratory methods for identification of CRAB carriers are not standardized. In this work, we compared different selective agar plates, tested the efficiency of A. baumannii identification by PCR for species-specific genes, and used PCR-based detection of common resistance genes to confirm the carbapenem-resistant phenotype. During a prospective study, we also determined the optimal sample enrichment time. Based on our results, we propose a simple and efficient protocol for the detection of CRAB carriage using skin sampling, short enrichment, selection on appropriate agar plates, and PCR-based identification, resulting in a turn-around time of 24 hours.

Active surveillance for carbapenem-resistant Acinetobacter baumannii (CRAB) carriage.

IMPORTANCE: Our study's results provide promising evidence for the incorporation of a high-sensitivity carbapenem-resistant Acinetobacter baumannii (CRAB) screening method in healthcare settings. Such an approach could prove beneficial in enhancing infection prevention and control measures, leading to improved patient outcomes and potentially alleviating the burden of CRAB in healthcare systems.

Analysis of four carbapenem-resistant Acinetobacter baumannii outbreaks using Fourier-transform infrared spectroscopy.

We used Fourier-transform infrared (FTIR) spectroscopy to analyze 4 carbapenem-resistant Acinetobacter baumannii outbreaks. FTIR distinguished between isolates from different hospitals and uncovered the relatedness between isolates from acute-care hospitals and a post-acute-care hospital. Using higher cutoffs reveals more distant relationships and lower cutoffs support analyses of recent events.

Occurrence, Typing, and Resistance Genes of ESBL/AmpC-Producing Enterobacterales in Fresh Vegetables Purchased in Central Israel.

Beta-lactam resistance can lead to increased mortality, higher healthcare expenses, and limited therapeutic options. The primary mechanism of beta-lactam resistance is the production of extended-spectrum beta-lactamases (ESBL) and AmpC beta-lactamases. The spread of beta-lactamase-producing Enterobacterales via the food chain may create a resistance reservoir. The aims of this study were to determine the prevalence of ESBL/AmpC-producing Enterobacterales in vegetables, to examine the association between EBSL/AmpC-producing bacteria and types of vegetables, packaging, and markets, and to investigate the genetic features of ESBL-producing isolates. The antibiotic susceptibilities were determined using VITEK. Phenotypic ESBL/AmpC production was confirmed using disk diffusion. ESBL-producing isolates were subjected to Fourier-transform infrared (FT-IR) spectroscopy and to whole genome sequencing using Oxford Nanopore sequencing technology. Of the 301 vegetable samples, 20 (6.6%) were positive for ESBL producers (16 Klebsiella pneumoniae and 4 Escherichia coli), and 63 (20.9%) were positive for AmpC producers (56 Enterobacter cloacae complex, 4 Enterobacter aerogenes/cancerogenus, and 3 Pantoea spp., Aeromonas hydrophila, and Citrobacter braakii). The blaCTX-M and blaSHV genes were most common among ESBL-producing isolates. The beta-lactamase genes of the ESBL producers were mainly carried on plasmids. Multilocus sequence typing and FT-IR typing revealed high diversity among the ESBL producers. AmpC producers were significantly more common in leafy greens and ESBL producers were significantly less common in climbing vegetables. The presence of ESBL/AmpC-producing Enterobacterales in raw vegetables may contribute to the dissemination of resistance genes in the community.

Phenotypic and Genomic Characterization of Nine String-Positive Carbapenem-Resistant Acinetobacter baumannii Isolates from Israel.

A positive "string test" indicates the ability of bacterial colonies grown on agar plates to form viscous strings of >5 mm when stretched. This phenotype is strongly associated with hypervirulence in Klebsiella pneumoniae but has never been described in carbapenem-resistant Acinetobacter baumannii (CRAB), an emerging human pathogen of high clinical significance. In this work, we screened 1,000 CRAB isolates, among which we identified and characterized 9 string-positive CRAB (stCRAB) isolates. Phenotypic and genotypic analyses revealed that the isolates were not phylogenetically related and possessed different antibiotic resistance and virulence profiles. Transmission electron microscopy (TEM) showed the presence of capsule in string-positive isolates. String-positive isolates were more motile but did not form more biofilm than non-string-positive isolates. They were less virulent in a murine thigh fitness model and a Galleria mellonella survival assay. In conclusion, here, we describe string-positive A. baumannii isolates and their phenotypic and molecular characteristics. We found that unlike K. pneumoniae, stCRAB isolates were not associated with increased virulence. IMPORTANCE Acinetobacter baumannii has been considered a major health care threat in recent years. Despite many efforts, the pathogenesis and molecular mechanism of A. baumannii virulence remain poorly understood. Moreover, the plasticity of its genome frequently gives rise to new and more virulent isolates. Our current study is of significant importance as it concerns a previously undescribed A. baumannii phenotype. The string-positive phenotype is strongly associated with increased fitness and virulence in other Gram-negative bacteria such as K. pneumoniae. Although no clear correlation with virulence or fitness was found in our 9 stCRAB isolates, this could have been due to the limited statistical power of our research. We suggest that this phenotype should be taken into consideration as due to its genome plasticity, the next change can give rise to string-positive and hypervirulent strains, as is known for K. pneumoniae. Additional future research is needed regarding its possible consequences.

Prevalence and Clinical Consequences of Colistin Heteroresistance and Evolution into Full Resistance in Carbapenem-Resistant Acinetobacter baumannii.

Colistin heteroresistance (HR) refers to a bacterial population comprised of several subpopulations with different levels of resistance to colistin. In this study, we discuss the classic form of HR, in which a resistant subpopulation exists within a predominantly susceptible population. We investigated the prevalence of colistin HR and its evolution into full resistance among 173 clinical carbapenem-resistant Acinetobacter baumannii isolates and examined the effect of HR on clinical outcomes. To determine HR, we performed population analysis profiling. Our results showed a high prevalence of HR (67.1%). To examine evolution of HR strains into full resistance, the HR strains were grown in colistin-containing broth, transferred onto colistin-containing plates, and colonies on these plates were transferred into colistin-free broth. Many of the HR strains (80.2%) evolved into full resistance, 17.2% reverted to HR, and 2.6% were borderline. We used logistic regression to compare 14-day clinical failure and 14-day mortality between patients infected by HR versus susceptible non-HR carbapenem-resistant A. baumannii. In the subgroup of patients with bacteremia, HR was significantly associated with 14-day mortality. IMPORTANCE To our knowledge, this is the first large-scale study to report on HR in Gram-negative bacteria. We described the prevalence of colistin HR in a large sample of carbapenem-resistant A. baumannii isolates, the evolution of many colistin HR isolates to a resistant phenotype following colistin exposure and withdrawal, and the clinical consequences of colistin HR. We found a high prevalence of HR among clinical carbapenem-resistant A. baumannii isolates; most evolved into a resistant phenotype following colistin exposure and withdrawal. In patients treated with colistin, evolution of HR A. baumannii into full resistance could lead to higher rates of treatment failure and contribute to the reservoir of colistin-resistant pathogens in health care settings.

Antizyme affects cell proliferation and viability solely through regulating cellular polyamines.

Antizymes are key regulators of cellular polyamine metabolism that negatively regulate cell proliferation and are therefore regarded as tumor suppressors. Although the regulation of antizyme (Az) synthesis by polyamines and the ability of Az to regulate cellular polyamine levels suggest the centrality of polyamine metabolism to its antiproliferative function, recent studies have suggested that antizymes might also regulate cell proliferation by targeting to degradation proteins that do not belong to the cellular polyamine metabolic pathway. Using a co-degradation assay, we show here that, although they efficiently stimulated the degradation of ornithine decarboxylase (ODC), Az1 and Az2 did not affect or had a negligible effect on the degradation of cyclin D1, Aurora-A, and a p73 variant lacking the N-terminal transactivation domain whose degradation was reported recently to be stimulated by Az1. Furthermore, we demonstrate that, although Az1 and Az2 could not be constitutively expressed in transfected cells, they could be stably expressed in cells that express trypanosome ODC, a form of ODC that does not bind Az and therefore maintains a constant level of cellular polyamines. Taken together, our results clearly demonstrate that Az1 and Az2 affect cell proliferation and viability solely by modulating cellular polyamine metabolism.

Unraveling the Diversity of Co-Colonization by CPE.

Antibiotic-resistant bacteria, and more specifically, carbapenem-producing Enterobacterales (CPE) strains, are increasing worldwide. Despite their growing prevalence, in most high-income countries, the detection of CPE is still considered a low-frequency event. Sporadically, patients co-colonized with distinct CPE strains and/or different carbapenemase enzymes are detected. In this paper, we present three cases that illustrate the underlying mechanisms of co-colonization, focusing on horizontal gene transfer (HGT) and patient-to-patient transmission. We also demonstrate the diversity of CPE species and discuss the potential consequences of co-colonization.

Calcium carbonate mineralization is essential for biofilm formation and lung colonization.

Biofilms are differentiated microbial communities held together by an extracellular matrix. µCT X-ray revealed structured mineralized areas within biofilms of lung pathogens belonging to two distant phyla - the proteobacteria Pseudomonas aeruginosa and the actinobacteria Mycobacterium abscessus. Furthermore, calcium chelation inhibited the assembly of complex bacterial structures for both organisms with little to no effect on cell growth. The molecular mechanisms promoting calcite scaffold formation were surprisingly conserved between the two pathogens as biofilm development was similarly impaired by genetic and biochemical inhibition of calcium uptake and carbonate accumulation. Moreover, chemical inhibition and mutations targeting mineralization significantly reduced the attachment of P. aeruginosa to the lung, as well as the subsequent damage inflicted by biofilms to lung tissues, and restored their sensitivity to antibiotics. This work offers underexplored druggable targets for antibiotics to combat otherwise untreatable biofilm infections.

The roles of intracellular and extracellular calcium in Bacillus subtilis biofilms.

In nature, bacteria reside in biofilms- multicellular differentiated communities held together by an extracellular matrix. This work identified a novel subpopulation-mineral-forming cells-that is essential for biofilm formation in Bacillus subtilis biofilms. This subpopulation contains an intracellular calcium-accumulating niche, in which the formation of a calcium carbonate mineral is initiated. As the biofilm colony develops, this mineral grows in a controlled manner, forming a functional macrostructure that serves the entire community. Consistently, biofilm development is prevented by the inhibition of calcium uptake. Our results provide a clear demonstration of the orchestrated production of calcite exoskeleton, critical to morphogenesis in simple prokaryotes.