Assessment of bacteriocin production by clinical Pseudomonas aeruginosa isolates and their potential as therapeutic agents.

INTRODUCTION: Bacterial infections and the rising antimicrobial resistance pose a significant threat to public health. Pseudomonas aeruginosa produces bacteriocins like pyocins, especially S-type pyocins, which are promising for biological applications. This research focuses on clinical P. aeruginosa isolates to assess their bacteriocin production, inhibitory spectrum, chemical structure, antibacterial agents, and preservative potential. METHODS: The identification of P. aeruginosa was conducted through both phenotypic and molecular approaches. The inhibitory spectrum and antibacterial potential of the isolates were assessed. The kinetics of antibacterial peptide production were investigated, and the activity of bacteriocin was quantified in arbitrary units (AU ml-1). Physico-chemical characterization of the antibacterial peptides was performed. Molecular weight estimation was carried out using SDS-PAGE. qRT-PCR analysis was employed to validate the expression of the selected candidate gene. RESULT: The antibacterial activity of P. aeruginosa was attributed to the secretion of bacteriocin compounds, which belong to the S-type pyocin family. The use of mitomycin C led to a significant 65.74% increase in pyocin production by these isolates. These S-type pyocins exhibited the ability to inhibit the growth of both Gram-negative (P. mirabilis and P. vulgaris) and Gram-positive (S. aureus, S. epidermidis, E. hirae, S. pyogenes, and S. mutans) bacteria. The molecular weight of S-type pyocin was 66 kDa, and its gene expression was confirmed through qRT-PCR. CONCLUSION: These findings suggest that S-type pyocin hold significant potential as therapeutic agents against pathogenic strains. The Physico-chemical resistance of S-type pyocin underscores its potential for broad applications in the pharmaceutical, hygiene, and food industries.

Therapeutic effect and anti-biofilm ability assessment of a novel phage, phiPA1-3, against carbapenem-resistant Pseudomonas aeruginosa.

Multiple drug-resistant (MDR) Pseudomonas aeruginosa commonly causes severe hospital-acquired infections. The gradual emergence of carbapenem-resistant P. aeruginosa has recently gained attention. A wide array of P. aeruginosa-mediated pathogenic mechanisms, including its biofilm-forming ability, limits the use of effective antimicrobial treatments against it. In the present study, we isolated and characterized the phenotypic, biological, and genomic characteristics of a bacteriophage, vB_PaP_phiPA1-3 (phiPA1-3). Biofilm eradication and phage rescue from bacterial infections were assessed to demonstrate the efficacy of the application potential. Host range spectrum analysis revealed that phiPA1-3 is a moderate host range phage that infects 20% of the clinically isolated strains of P. aeruginosa tested, including carbapenem-resistant P. aeruginosa (CRPA). The phage exhibited stability at pH 7.0 and 9.0, with significantly reduced viability below pH 5.0 and beyond pH 9.0. phiPA1-3 is a lytic phage with a burst size of 619 plaque-forming units/infected cell at 37 °C and can effectively lyse bacteria in a multiplicity of infection-dependent manner. The genome size of phiPA1-3 was found to be 73,402 bp, with a G+C content of 54.7%, containing 93 open reading frames, of which 62 were annotated as hypothetical proteins and the remaining 31 had known functions. The phage possesses several proteins similar to those found in N4-like phages, including three types of RNA polymerases. This study concluded that phiPA1-3 belongs to the N4-like Schitoviridae family, can potentially eradicate P. aeruginosa biofilms, and thus, serve as a valuable tool for controlling CRPA infections.

Directed carbapenemase testing is no longer just for Enterobacterales: cost, labor, and workflow assessment of expanding carbapenemase testing to carbapenem-resistant P. aeruginosa.

Molecular carbapenem-resistance testing, such as for the presence of carbapenemases genes, is commonly implemented for the detection of carbapenemase-producing Enterobacterales. Carbapenemase-producing P. aeruginosa is also associated with significant morbidity and mortality, although; prevalence may be underappreciated in the United States due to a lack of carbapenemase testing. The present study sought to compare hands-on time, cost and workflow implementation of carbapenemase gene testing in Enterobacterales and P. aeruginosa isolates versus sending out isolates to a public health laboratory (PHL) for testing to assess if in-house can provide actionable results. The time to carbapenemase gene results were compared. Differences in cost for infection prevention measures were extrapolated from the time of positive carbapenemase gene detection in-house versus PHL. The median time to perform carbapenemase gene testing was 7.5 min (range 5-14) versus 10 min (range 8-22) for preparation to send isolates to the PHL. In-house testing produced same day results compared with a median of 6 days (range 3-14) to receive results from PHL. Cost of in-house testing and send outs were similar ($46.92 versus $40.53, respectively). If contact precautions for patients are implemented until carbapenemase genes are ruled out, in-house testing can save an estimated $76,836.60 annually. Extension of in-house carbapenemase testing to include P. aeruginosa provides actionable results 3-14 days earlier than PHL Standard Pathway testing, facilitating guided therapeutic decisions and infection prevention measures. Supplemental phenotypic algorithms can be implemented to curb the cost of P. aeruginosa carbapenemases testing by identifying isolates most likely to harbour carbapenemases.

Carbapenem-resistant Pseudomonas aeruginosa: an assessment of frequency of isolation from ICU versus non-ICU, phenotypic and genotypic profiles in a multinational population of hospitalized patients.

BACKGROUND: Historically, multi-drug resistant organisms have been associated with the ICU setting. The present study sought to define the frequency of isolation from ICU versus non-ICU, phenotypic and genotypic profiles of carbapenem-resistant Pseudomonas aeruginosa (CR-PA) from a global cohort. METHODS: Multicenter surveillance study (17 centers from 12 countries) including 672 CR-PA isolates from 2019 to 2021. Phenotypic carbapenemase testing was assessed. Genotypic carbapenemase testing was conducted (CarbaR and CarbaR NxG) to detect ß-lactamases. Broth microdilution MICs were established for ceftazidime, cefepime, ceftolozane/tazobactam, and ceftazidime/avibactam. RESULTS: 59% of CR-PA were isolated from patients outside the ICU. The most common source in ICU and non-ICU patients was respiratory (55% and 30%, respectively). In the ICU, 35% of isolates were phenotypically carbapenemase-positive versus 29% for non-ICU. VIM was the most common carbapenemase (54% and 44%, respectively) followed by GES (27% and 28%, respectively). Susceptibility to ceftazidime or cefepime were relatively low in ICU (39% and 41% of isolates, respectively) and non-ICU (47% and 52% of isolates, respectively). Ceftolozane/tazobactam and ceftazidime/avibactam were more active with 56% and 66% of isolates susceptible in the ICU while 65% and 76% in non-ICU, respectively. When carbapenemase-negative, 86% and 88% of ICU isolates were susceptible to ceftolozane/tazobactam and ceftazidime/avibactam. Similarly, in the carbapenemase-negative, non-ICU isolates 88% and 92% of isolates were susceptible, respectively. CONCLUSION: Although multidrug resistant pathogens are often regarded as a challenge in the ICU population, the majority of CR-PA were isolated from non-ICU patients. Implementing phenotypic/genotypic testing will assist in guiding treatment. Carbapenem-resistance in P. aeruginosa should be regarded as a surrogate for MDR and this phenotype is increasingly prevalent outside the ICU.

Role of Enzymatic Activity in the Biological Cost Associated with the Production of AmpC ß-Lactamases in Pseudomonas aeruginosa.

In the current scenario of growing antibiotic resistance, understanding the interplay between resistance mechanisms and biological costs is crucial for designing therapeutic strategies. In this regard, intrinsic AmpC ß-lactamase hyperproduction is probably the most important resistance mechanism of Pseudomonas aeruginosa, proven to entail important biological burdens that attenuate virulence mostly under peptidoglycan recycling alterations. P. aeruginosa can acquire resistance to new ß-lactam-ß-lactamase inhibitor combinations (ceftazidime-avibactam and ceftolozane-tazobactam) through mutations affecting ampC and its regulatory genes, but the impact of these mutations on the associated biological cost and the role that ß-lactamase activity plays per se in contributing to the above-mentioned virulence attenuation are unknown. The same questions remain unsolved for plasmid-encoded AmpC-type ß-lactamases such as FOX enzymes, some of which also provide resistance to new ß-lactam-ß-lactamase inhibitor combinations. Here, we assessed from different perspectives the effects of changes in the active center and, thus, in the hydrolytic spectrum resistance to inhibitors of AmpC-type ß-lactamases on the fitness and virulence of P. aeruginosa, using site-directed mutagenesis; the previously described AmpC variants T96I, G183D, and ΔG229-E247; and, finally, blaFOX-4 versus blaFOX-8. Our results indicate the essential role of AmpC activity per se in causing the reported full virulence attenuation (in terms of growth, motility, cytotoxicity, and Galleria mellonella larvae killing), although the biological cost of the above-mentioned AmpC-type variants was similar to that of the wild-type enzymes. This suggests that there is not an important biological burden that may limit the selection/spread of these variants, which could progressively compromise the future effectiveness of the above-mentioned drug combinations. IMPORTANCE The growing antibiotic resistance of the top nosocomial pathogen Pseudomonas aeruginosa pushes research to explore new therapeutic strategies, for which the resistance-versus-virulence balance is a promising source of targets. While resistance often entails significant biological costs, little is known about the bases of the virulence attenuations associated with a resistance mechanism as extraordinarily relevant as ß-lactamase production. We demonstrate that besides potential energy and cell wall alterations, the enzymatic activity of the P. aeruginosa cephalosporinase AmpC is essential for causing the full attenuation associated with its hyperproduction by affecting different features related to pathogenesis, a fact exploitable from the antivirulence perspective. Less encouraging, we also show that the production of different chromosomal/plasmid-encoded AmpC derivatives conferring resistance to some of the newest antibiotic combinations causes no significantly increased biological burdens, which suggests a free way for the selection/spread of these types of variants, potentially compromising the future effectiveness of these antipseudomonal therapies.

In vivo translational assessment of the GES genotype on the killing profile of ceftazidime, ceftazidime/avibactam and meropenem against Pseudomonas aeruginosa.

OBJECTIVES: To evaluate the in vivo killing profile of human-simulated exposures of ceftazidime, ceftazidime/avibactam and meropenem against GES-harbouring Pseudomonas aeruginosa in the murine thigh infection model. METHODS: Five P. aeruginosa isolates [three isogenic (GES-1, GES-5 and GES-15) and two clinical (GES-5 and GES-15)] were evaluated. MICs were determined using broth microdilution. Human-simulated regimens (HSRs) of ceftazidime 2 g IV q8h as a 2 h infusion, ceftazidime/avibactam 2.5 g IV q8h as a 2 h infusion and meropenem 2 g IV q8h as a 3 h infusion were administered. Change in bacterial burden relative to baseline was assessed. RESULTS: Modal MICs ranged from 8 to >64 mg/L for ceftazidime, from 1 to 16 mg/L for ceftazidime/avibactam and from 1 to >64 mg/L for meropenem. In vivo, for the isogenic strains, avibactam augmented ceftazidime activity against the GES-1- and GES-15-harbouring isolates. Both ceftazidime and ceftazidime/avibactam resulted in significant kill against the GES-5 isogenic isolate. The meropenem HSR produced >1 log10 kill against each isogenic isolate (MICs of 1-4 mg/L). Against the GES-5 clinical isolate, ceftazidime and ceftazidime/avibactam resulted in >1 log10 kill compared with bacterial growth with the meropenem HSR. In the clinical isolate harbouring GES-15, the elevated MICs of ceftazidime and ceftazidime/avibactam reduced the effectiveness of both compounds, while the observed reduction in meropenem MIC translated into in vivo efficacy of the HSR regimen, predictive of clinical efficacy. CONCLUSIONS: In GES-harbouring P. aeruginosa, quantitative reductions in bacterial density observed with the translational murine model suggest that the phenotypic profile of ceftazidime, ceftazidime/avibactam and meropenem is predictive of clinical efficacy when using the evaluated dosing regimens.

Histological assessment, anti-quorum sensing, and anti-biofilm activities of Dioon spinulosum extract: in vitro and in vivo approach.

Pseudomonas aeruginosa is an opportunistic bacterium causing several health problems and having many virulence factors like biofilm formation on different surfaces. There is a significant need to develop new antimicrobials due to the spreading resistance to the commonly used antibiotics, partly attributed to biofilm formation. Consequently, this study aimed to investigate the anti-biofilm and anti-quorum sensing activities of Dioon spinulosum, Dyer Ex Eichler extract (DSE), against Pseudomonas aeruginosa clinical isolates. DSE exhibited a reduction in the biofilm formation by P. aeruginosa isolates both in vitro and in vivo rat models. It also resulted in a decrease in cell surface hydrophobicity and exopolysaccharide quantity of P. aeruginosa isolates. Both bright field and scanning electron microscopes provided evidence for the inhibiting ability of DSE on biofilm formation. Moreover, it reduced violacein production by Chromobacterium violaceum (ATCC 12,472). It decreased the relative expression of 4 quorum sensing genes (lasI, lasR, rhlI, rhlR) and the biofilm gene (ndvB) using qRT-PCR. Furthermore, DSE presented a cytotoxic activity with IC50 of 4.36 ± 0.52 µg/ml against human skin fibroblast cell lines. For the first time, this study reports that DSE is a promising resource of anti-biofilm and anti-quorum sensing agents.

Hackflex: low-cost, high-throughput, Illumina Nextera Flex library construction.

We developed a low-cost method for the production of Illumina-compatible sequencing libraries that allows up to 14 times more libraries for high-throughput Illumina sequencing to be generated for the same cost. We call this new method Hackflex. The quality of library preparation was tested by constructing libraries from Escherichia coli MG1655 genomic DNA using either Hackflex, standard Nextera Flex (recently renamed as Illumina DNA Prep) or a variation of standard Nextera Flex in which the bead-linked transposase is diluted prior to use. In order to test the library quality for genomes with a higher and a lower G+C content, library construction methods were also tested on Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC 25923, respectively. We demonstrated that Hackflex can produce high-quality libraries and yields a highly uniform coverage, equivalent to the standard Nextera Flex kit. We show that strongly size-selected libraries produce sufficient yield and complexity to support de novo microbial genome assembly, and that assemblies of the large-insert libraries can be much more contiguous than standard libraries without strong size selection. We introduce a new set of sample barcodes that are distinct from standard Illumina barcodes, enabling Hackflex samples to be multiplexed with samples barcoded using standard Illumina kits. Using Hackflex, we were able to achieve a per-sample reagent cost for library prep of A$7.22 (Australian dollars) (US $5.60; UK £3.87, £1=A$1.87), which is 9.87 times lower than the standard Nextera Flex protocol at advertised retail price. An additional simple modification and further simplification of the protocol by omitting the wash step enables a further price reduction to reach an overall 14-fold cost saving. This method will allow researchers to construct more libraries within a given budget, thereby yielding more data and facilitating research programmes where sequencing large numbers of libraries is beneficial.

A low-cost, portable, and practical LAMP device for point-of-diagnosis in the field.

Transition of rapid, ready-to-use, and low-cost nucleic acid-based detection technologies from laboratories to points of sample collection has drastically accelerated. However, most of these approaches are still incapable of diagnosis starting from sampling through nucleic acid isolation and detection in the field. Here we developed a simple, portable, low-cost, colorimetric, and remotely controllable platform for reliable, high-throughput, and rapid diagnosis using loop-mediated isothermal amplification (LAMP) assays. It consists of a thermally isolated cup, low-cost electronic components, a polydimethylsiloxane sample well, and a fast prototyped case that covers electronic components. The steady-state temperature error of the system is <1%. We performed LAMP, Colony-LAMP, and Colony polymerase chain reactions (PCRs) using the yaiO2 primer set for Escherichia coli and Pseudomonas aeruginosa samples at 65°C and 30 min. We detected the end-point colorimetric readouts by the naked eye under day light. We confirmed the specificity and sensitivity of our approach using pure genomic DNA and crude bacterial colonies. We benchmarked our Colony-LAMP detection against Colony PCR. The number of samples tested can easily be modified for higher throughput in our system. We strongly believe that our platform can greatly contribute rapid and reliable diagnosis in versatile operational environments.

Outbreak of Pseudomonas aeruginosa producing VIM carbapenemase in an intensive care unit and its termination by implementation of waterless patient care.

BACKGROUND: Long-term outbreaks of multidrug-resistant Gram-negative bacilli related to hospital-building water systems have been described. However, successful mitigation strategies have rarely been reported. In particular, environmental disinfection or replacement of contaminated equipment usually failed to eradicate environmental sources of Pseudomonas aeruginosa. METHODS: We report the investigation and termination of an outbreak of P. aeruginosa producing VIM carbapenemase (PA-VIM) in the adult intensive care unit (ICU) of a Swiss tertiary care hospital with active case finding, environmental sampling and whole genome sequencing (WGS) of patient and environmental strains. We also describe the implemented control strategies and their effectiveness on eradication of the environmental reservoir. RESULTS: Between April 2018 and September 2020, 21 patients became either infected or colonized with a PA-VIM strain. For 16 of them, an acquisition in the ICU was suspected. Among 131 environmental samples collected in the ICU, 13 grew PA-VIM in sink traps and drains. WGS confirmed the epidemiological link between clinical and environmental strains and the monoclonal pattern of the outbreak. After removing sinks from patient rooms and implementation of waterless patient care, no new acquisition was detected in the ICU within 8 months after the intervention. DISCUSSION: Implementation of waterless patient care with removal of the sinks in patient rooms was successful for termination of a PA-VIM ICU outbreak linked to multiple environmental water sources. WGS provides highly discriminatory accuracy to investigate environment-related outbreaks.

Phage gene expression and host responses lead to infection-dependent costs of CRISPR immunity.

CRISPR-Cas immune systems are widespread in bacteria and archaea, but not ubiquitous. Previous work has demonstrated that CRISPR immunity is associated with an infection-induced fitness cost, which may help explain the patchy distribution observed. However, the mechanistic basis of this cost has remained unclear. Using Pseudomonas aeruginosa PA14 and its phage DMS3vir as a model, we perform a 30-day evolution experiment under phage mediated selection. We demonstrate that although CRISPR is initially selected for, bacteria carrying mutations in the phage receptor rapidly invade the population following subsequent reinfections. We then test three potential mechanisms for the observed cost of CRISPR: (1) autoimmunity from the acquisition of self-targeting spacers, (2) immunopathology or energetic costs from increased cas gene expression and (3) toxicity caused by phage gene expression prior to CRISPR-mediated cleavage. We find that phages can express genes before the immune system clears the infection and that expression of these genes can have a negative effect on host fitness. While infection does not lead to increased expression of cas genes, it does cause differential expression of multiple other host processes that may further contribute to the cost of CRISPR immunity. In contrast, we found little support for infection-induced autoimmunological and immunopathological effects. Phage gene expression prior to cleavage of the genome by the CRISPR-Cas immune system is therefore the most parsimonious explanation for the observed phage-induced fitness cost.

Low nutrient levels as drinking water conditions can reduce the fitness cost of efflux pump-mediated ciprofloxacin resistance in Pseudomonas aeruginosa.

The long-term persistence of antibiotic resistance in the environment, especially in drinking water, is a public health concern. Expression of an efflux pump, an important mechanism of resistance to antibiotics, usually confers a fitness cost in bacteria. In this study, we aimed to determine why antibiotic resistance conferred by overexpression of an efflux pump persisted in low-nutrient environments (TOC < 10 mg/L) such as drinking and source water in which antibiotic selective pressure might be very low or even absent. Competition experiments between wild-type Pseudomonas aeruginosa and ciprofloxacin-resistant mutants revealed that the fitness cost of ciprofloxacin resistance significantly decreased (p < 0.05) under low-nutrient (0.5 mg/L total organic carbon (TOC)) relative to high-nutrient (500 mg/L TOC) conditions. Mechanisms underlying this fitness cost were analyzed. The mexD gene expression in resistant bacteria (cip_3 strain) was significantly lower (p < 0.05) in low-nutrient conditions, with 10 mg/L TOC ((8.01 ±â€¯0.82)-fold), than in high-nutrient conditions, with 500 mg/L TOC ((48.89 ±â€¯4.16)-fold). Moreover, rpoS gene expression in resistant bacteria ((1.36 ±â€¯0.13)-fold) was significantly lower (p < 0.05) than that in the wild-type strain ((2.78 ±â€¯0.29)-fold) under low-nutrient conditions (10 mg/L TOC), suggesting a growth advantage. Furthermore, the difference in metabolic activity between the two competing strains was significantly smaller (p < 0.05) in low-nutrient conditions (5 and 0.5 mg/L TOC). These results suggest that nutrient levels are a key factor in determining the persistence of antibiotic resistance conferred by efflux pumps in the natural environment with trace amounts or no antibiotics.

Mortality related to Verona Integron-encoded Metallo-ß-lactamase-positive Pseudomonas aeruginosa: assessment by a novel clinical tool.

Background: Verona Integron-encoded Metallo-ß-lactamase-positive Pseudomonas aeruginosa (VIM-PA) can cause nosocomial infections and may be responsible for increased mortality. Multidrug resistance in VIM-PA complicates treatment. We aimed to assess the contribution of VIM-PA to mortality in patients in a large tertiary care hospital in the Netherlands. Methods: A focus group of five members created a scheme to define related mortality based on clinical and diagnostic findings. Contribution to mortality was categorized as "definitely", "probably", "possibly", or "not" related to infection with VIM-PA, or as "unknown". Patients were included when infected with or carrier of VIM-PA between January 2008 and January 2016. Patient-related data and specific data on VIM-PA cultures were retrieved from the electronic laboratory information system. For patients who died in our hospital, medical records were independently reviewed and thereafter discussed by three physicians. Results: A total of 198 patients with any positive culture with VIM-PA were identified, of whom 95 (48.0%) died. Sixty-seven patients died in our hospital and could be included in the analysis. The death of 15 patients (22.4%) was judged by all reviewers to be definitely related to infection with VIM-PA. In 17 additional patients (25.4%), death was probably or possibly related to an infection with VIM-PA. The level of agreement was 65.7% after the first evaluation, and 98.5% after one session of discussion. Conclusion: Using our assessment tool, infections with VIM-PA were shown to have an important influence on mortality in our complex and severely ill patients. The tool may be used for other (resistant) bacteria as well but this needs further exploration.

Assessment of biofilm formation by pseudomonas aeruginosa and hydrodynamic evaluation of microtiter plate assay.

OBJECTIVE: To assess the biofilm formation in clinical and environmental isolates of Pseudomonas aeruginosa and to evaluate the hydrodynamics in microtiter plate assay and compare it with conventional assays for biofilm formation. METHODS: The cross-sectional study was conducted at the Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan, in 2013-14, while the computational work was done at the National University of Science and Technology, Islamabad. The study comprised environmental and clinical isolates of pseudomonas aeruginosa. Pseudomonas citramide agar was used as a selective media, and further confirmation was done by biochemical tests. Biofilm formation was assessed by Congo red assay, air liquid interfaceassay and microtiter plate assay. Computational Fluid Dynamics (CFD) simulations were also used to improve the microtiter plate assay for biofilm formation assessment. Polymerase chain reaction was used for screening of pelA and pelG genes. RESULTS: Of the 50 isolates, 25(50%) each were environmental and clinical. The number of biofilm producers observed in Congo red assay, air liquid interface assay and microtiter plate assay were 7(14%), 15(30%) and 30(60%) respectively. Biofilm former gene pelA was observed in 22(44%) isolates while 36(72%) isolates showed the presence of pelG gene. CONCLUSIONS: Microtiter plate assay was found to be a reliable method to detect biofilm forming pseudomonas aeruginosa isolates which further provides a base for development of methods to detect biofilms readily and accurately.

Fast, economic and simultaneous identification of clinically relevant Gram-negative species with multiplex real-time PCR.

AIM: A newly designed multiplex real-time PCR (rt-PCR) was validated to detect four clinically relevant Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa). MATERIALS & METHODS: Serial dilutions of genomic DNA were used to determine the limit of detection. Colony PCR was performed with isolates of the four selected species and other species as negative controls. Isolates were characterized genotypically and phenotypically to evaluate the assay. RESULTS: Specific signals of all target genes were detected with diluted templates comprising ten genomic equivalents. Using colony rt-PCR, all isolates of the target species were identified correctly. All negative control isolates were negative. CONCLUSION: The genes gad, basC, khe and ecfX can reliably identify these four species via multiplex colony rt-PCR.

Buckyballs conjugated with nucleic acid sequences identifies microorganisms in live cell assays.

BACKGROUND: Rapid identification of bacteria can play an important role at the point of care, evaluating the health of the ecosystem, and discovering spatiotemporal distributions of a bacterial community. We introduce a method for rapid identification of bacteria in live cell assays based on cargo delivery of a nucleic acid sequence and demonstrate how a mixed culture can be differentiated using a simple microfluidic system. METHODS: C60 Buckyballs are functionalized with nucleic acid sequences and a fluorescent reporter to show that a diversity of microorganisms can be detected and identified in live cell assays. The nucleic acid complexes include an RNA detector, targeting a species-specific sequence in the 16S rRNA, and a complementary DNA with an attached fluorescent reporter. As a result, each bacterium can be detected and visualized at a specific emission frequency through fluorescence microscopy. RESULTS: The C60 probe complexes can detect and identify a diversity of microorganisms that include gram-position and negative bacteria, yeast, and fungi. More specifically, nucleic-acid probes are designed to identify mixed cultures of Bacillus subtilis and Streptococcus sanguinis, or Bacillus subtilis and Pseudomonas aeruginosa. The efficiency, cross talk, and accuracy for the C60 probe complexes are reported. Finally, to demonstrate that mixed cultures can be separated, a microfluidic system is designed that connects a single source-well to multiple sinks wells, where chemo-attractants are placed in the sink wells. The microfluidic system allows for differentiating a mixed culture. CONCLUSIONS: The technology allows profiling of bacteria composition, at a very low cost, for field studies and point of care.

Diversity and distribution of nuclease bacteriocins in bacterial genomes revealed using Hidden Markov Models.

Bacteria exploit an arsenal of antimicrobial peptides and proteins to compete with each other. Three main competition systems have been described: type six secretion systems (T6SS); contact dependent inhibition (CDI); and bacteriocins. Unlike T6SS and CDI systems, bacteriocins do not require contact between bacteria but are diffusible toxins released into the environment. Identified almost a century ago, our understanding of bacteriocin distribution and prevalence in bacterial populations remains poor. In the case of protein bacteriocins, this is because of high levels of sequence diversity and difficulties in distinguishing their killing domains from those of other competition systems. Here, we develop a robust bioinformatics pipeline exploiting Hidden Markov Models for the identification of nuclease bacteriocins (NBs) in bacteria of which, to-date, only a handful are known. NBs are large (>60 kDa) toxins that target nucleic acids (DNA, tRNA or rRNA) in the cytoplasm of susceptible bacteria, usually closely related to the producing organism. We identified >3000 NB genes located on plasmids or on the chromosome from 53 bacterial species distributed across different ecological niches, including human, animals, plants, and the environment. A newly identified NB predicted to be specific for Pseudomonas aeruginosa (pyocin Sn) was produced and shown to kill P. aeruginosa thereby validating our pipeline. Intriguingly, while the genes encoding the machinery needed for NB translocation across the cell envelope are widespread in Gram-negative bacteria, NBs are found exclusively in γ-proteobacteria. Similarity network analysis demonstrated that NBs fall into eight groups each with a distinct arrangement of protein domains involved in import. The only structural feature conserved across all groups was a sequence motif critical for cell-killing that is generally not found in bacteriocins targeting the periplasm, implying a specific role in translocating the nuclease to the cytoplasm. Finally, we demonstrate a significant association between nuclease colicins, NBs specific for Escherichia coli, and virulence factors, suggesting NBs play a role in infection processes, most likely by enabling pathogens to outcompete commensal bacteria.

Metabolic Compensation of Fitness Costs Is a General Outcome for Antibiotic-Resistant Pseudomonas aeruginosa Mutants Overexpressing Efflux Pumps.

It is generally assumed that the acquisition of antibiotic resistance is associated with a fitness cost. We have shown that overexpression of the MexEF-OprN efflux pump does not decrease the fitness of a resistant Pseudomonas aeruginosa strain compared to its wild-type counterpart. This lack of fitness cost was associated with a metabolic rewiring that includes increased expression of the anaerobic nitrate respiratory chain when cells are growing under fully aerobic conditions. It was not clear whether this metabolic compensation was exclusive to strains overexpressing MexEF-OprN or if it extended to other resistant strains that overexpress similar systems. To answer this question, we studied a set of P. aeruginosa mutants that independently overexpress the MexAB-OprM, MexCD-OprJ, or MexXY efflux pumps. We observed increased expression of the anaerobic nitrate respiratory chain in all cases, with a concomitant increase in NO3 consumption and NO production. These efflux pumps are proton/substrate antiporters, and their overexpression may lead to intracellular H+ accumulation, which may in turn offset the pH homeostasis. Indeed, all studied mutants showed a decrease in intracellular pH under anaerobic conditions. The fastest way to eliminate the excess of protons is by increasing oxygen consumption, a feature also displayed by all analyzed mutants. Taken together, our results support metabolic rewiring as a general mechanism to avoid the fitness costs derived from overexpression of P. aeruginosa multidrug efflux pumps. The development of drugs that block this metabolic "reaccommodation" might help in reducing the persistence and spread of antibiotic resistance elements among bacterial populations.IMPORTANCE It is widely accepted that the acquisition of resistance confers a fitness cost in such a way that in the absence of antibiotics, resistant populations will be outcompeted by susceptible ones. Based on this assumption, antibiotic cycling regimes have been proposed in the belief that they will reduce the persistence and spread of resistance among bacterial pathogens. Unfortunately, trials testing this possibility have frequently failed, indicating that resistant microorganisms are not always outcompeted by susceptible ones. Indeed, some mutations do not result in a fitness cost, and in case they do, the cost may be compensated for by a secondary mutation. Here we describe an alternative nonmutational mechanism for compensating for fitness costs, which consists of the metabolic rewiring of resistant mutants. Deciphering the mechanisms involved in the compensation of fitness costs of antibiotic-resistant mutants may help in the development of drugs that will reduce the persistence of resistance by increasing said costs.

A novel GoldNano Carb test for rapid phenotypic detection of carbapenemases, particularly OXA type, in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp.

Objectives: To develop a simple gold nanoparticle (AuNP)-based colorimetric test, GoldNano Carb (GoldC), for detecting carbapenemase production in Gram-negative bacteria, compared with updated Carba NP (CNP) and CarbAcineto NP (CAcNP) tests by using PCR methods as gold standard. Methods: Ninety-nine carbapenemase-producing Enterobacteriaceae (CPE), Pseudomonas spp. and Acinetobacter spp. isolates and 89 non-CPE isolates were tested by the GoldC and CNP. Additionally, the CAcNP was performed in the Acinetobacter spp. isolates. The final imipenem (imipenem/cilastatin form) concentration was 5 mg/mL for all three tests. For the GoldC, the imipenem powder was added directly to bacterial cell suspension in distilled water prior to detection of acid product by the citrate-capped AuNP solution. An AuNP change from red to purple, blue or green indicates carbapenemase activity. Results: The GoldC detected all carbapenemase producers except one OXA-23-like producer (99.0% sensitivity), whereas 11 carbapenemase producers (10 Acinetobacter and 1 P. aeruginosa) were CNP negative (88.9% sensitivity). However, the GoldC and CNP provided 100% and 98.6% sensitivity, respectively, for the CPE and Pseudomonas spp. Both tests gave one false positive from CTX-M-1-like-producing Enterobacter spp. (98.9% specificity). The GoldC and CAcNP detected 96.7% and 93.3% of the Acinetobacter spp. isolates, respectively. Interestingly, times to positivity by the GoldC were markedly shorter than those by the CNP (76.8% versus 36.2% positive at 5 min) and CAcNP (43.3% at 5 min versus 20% within 30 min). Conclusions: The GoldC is fast, easy, highly sensitive and inexpensive (∼$0.25 per test), suggesting that it may be suitable for routine carbapenemase detection in low-resource settings for infection control or epidemiological purposes.

Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules.

Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope.