The Use of CHROMID® Colistin R for the Detection of Colistin-Resistant Gram-Negative Bacteria in Positive Blood Cultures.

The aim of this study was to assess the utility of CHROMID® Colistin R for direct detection of colistin-resistant Gram-negative bacteria from positive blood cultures. A total of 390 blood cultures from hospitalised patients containing Gram-negative bacteria were included in this study. These blood cultures were referred to clinical laboratories in the United Kingdom and Türkiye. A further 16 simulated positive blood culture bottles were included that contained a range of colistin-resistant strains as well as susceptible control strains. Fluid from each positive blood culture was diluted 1/200 in saline and 10 µL aliquots cultured onto cystine-lactose-electrolyte-deficient agar and CHROMID® Colistin R. All recovered bacteria were identified, and for Gram-negative bacteria, their minimum inhibitory concentration of colistin was measured using the broth microdilution method. From a total of 443 Gram-negative isolates, 57 colistin-resistant isolates were recovered, of which 53 (93%) grew on CHROMID® Colistin R within 18 h. Of the 377 isolates determined to be colistin-susceptible, only 9 isolates were able to grow, including 6 isolates of Pseudomonas aeruginosa. For positive blood cultures that are shown to contain Gram-negative bacteria, culture on CHROMID® Colistin R is a useful diagnostic tool to detect susceptibility or resistance to colistin within 18 h.

Chromogenic enzyme substrates based on [2-(nitroaryl)ethenyl]pyridinium and quinolinium derivatives for the detection of nitroreductase activity in clinically important microorganisms.

A series of [2-(nitroaryl)ethenyl]pyridinium and quinolinium derivatives have been synthesised as potential indicators of microbial nitroreductase activity. When assessed against a selection of 20 clinically important pathogenic microorganisms, microbial colonies of various colours (yellow, green, red, brown, black) were produced and attributed to nitroreductase activity. Most substrates elicited colour responses with Gram-negative microorganisms. In contrast, the growth of several species of Gram-positive microorganisms and yeasts was often inhibited by the substrates and hence coloured responses were not seen.

Evaluation of a Novel Chromogenic Medium for the Detection of Pseudomonas aeruginosa in Respiratory Samples from Patients with Cystic Fibrosis.

Pseudomonas aeruginosa is a dominant cause of respiratory infection in individuals with cystic fibrosis (CF), leading to significant morbidity and mortality. Detection of P. aeruginosa is conducted by culture of respiratory samples but this process may occasionally be compromised due to overgrowth by other bacteria and fungi. We aimed to evaluate a novel chromogenic medium, Pseudomonas aeruginosa chromogenic agar (PACA), for culture of P. aeruginosa from respiratory samples, from patients with CF. A total of 198 respiratory samples were cultured onto PACA and three other media: CHROMID® P. aeruginosa, CHROMagar™ Pseudomonas and MacConkey agar. P. aeruginosa was recovered from 66 samples (33%), using a combination of all media. After 72 h incubation, the sensitivity of the four chromogenic media was as follows: 91% for PACA and CHROMagar™ Pseudomonas, 85% for CHROMID® P. aeruginosa and 83% for MacConkey agar. For the three chromogenic media, the positive predictive value after 72 h was as follows: 95% for PACA, 56% for CHROMagar™ Pseudomonas and 86% for CHROMID® P. aeruginosa. PACA proved to be a highly effective culture medium for the isolation and specific detection of P. aeruginosa from respiratory samples.

Contemporary diagnostics for medically relevant fastidious microorganisms belonging to the genera Anaplasma,Bartonella,Coxiella,OrientiaandRickettsia.

Many of the human infectious pathogens-especially the zoonotic or vector-borne bacteria-are fastidious organisms that are difficult to cultivate because of their strong adaption to the infected host culminating in their near-complete physiological dependence on this environment. These bacterial species exhibit reduced multiplication rates once they are removed from their optimal ecological niche. This fact complicates the laboratory diagnosis of the disease and hinders the detection and further characterization of the underlying organisms, e.g. at the level of their resistance to antibiotics due to their slow growth. Here, we describe the current state of microbiological diagnostics for five genera of human pathogens with a fastidious laboratory lifestyle. For Anaplasma spp., Bartonella spp., Coxiella burnetii, Orientia spp. and Rickettsia spp., we will summarize the existing diagnostic protocols, the specific limitations for implementation of novel diagnostic approaches and the need for further optimization or expansion of the diagnostic armamentarium. We will reflect upon the diagnostic opportunities provided by new technologies including mass spectrometry and next-generation nucleic acid sequencing. Finally, we will review the (im)possibilities of rapidly developing new in vitro diagnostic tools for diseases of which the causative agents are fastidiously growing and therefore hard to detect.

Recent Advances in Rapid Antimicrobial Susceptibility Testing.

BACKGROUND: Antimicrobial susceptibility testing (AST) is classically performed using growth-based techniques that essentially require viable bacterial matter to become visible to the naked eye or a sophisticated densitometer. CONTENT: Technologies based on the measurement of bacterial density in suspension have evolved marginally in accuracy and rapidity over the 20th century, but assays expanded for new combinations of bacteria and antimicrobials have been automated, and made amenable to high-throughput turn-around. Over the past 25 years, elevated AST rapidity has been provided by nucleic acid-mediated amplification technologies, proteomic and other "omic" methodologies, and the use of next-generation sequencing. In rare cases, AST at the level of single-cell visualization was developed. This has not yet led to major changes in routine high-throughput clinical microbiological detection of antimicrobial resistance. SUMMARY: We here present a review of the new generation of methods and describe what is still urgently needed for their implementation in day-to-day management of the treatment of infectious diseases.

Automated antimicrobial susceptibility testing of slow-growing Pseudomonas aeruginosa strains in the presence of tetrazolium salt WST-1.

Slow growing, mucoid isolates of Pseudomonas aeruginosa require adaptation of the protocol used for automated antimicrobial susceptibility testing (AST). In the present study we used a water soluble tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) in combination with menadione for possibly improving AST of slow growing and biofilm-forming P. aeruginosa isolates from cystic fibrosis (CF) patients. WST-1 and menadione addition ensures sensitive detection of microbial growth increase in the presence of antibiotics that may remain undetected with the automated VITEK® 2 method. We observed that 32.8% of P. aeruginosa isolates from CF and bronchiectasis patients produced an elevated absorbance signal intensity thereby increasing the sensitivity while maintaining the accuracy of VITEK 2. Our study merits future investigation with other slow growing pathogenic bacterial species.

Phenotypic and Genomic Variability of Serial Peri-Lung Transplantation Pseudomonas aeruginosa Isolates From Cystic Fibrosis Patients.

Cystic fibrosis (CF) represents one of the major genetic and chronic lung diseases affecting Caucasians of European descent. Patients with CF suffer from recurring infections that lead to further damage of the lungs. Pulmonary infection due to Pseudomonas aeruginosa is most prevalent, further increasing CF-related mortality. The present study describes the phenotypic and genotypic variations among 36 P. aeruginosa isolates obtained serially from a non-CF and five CF patients before, during and after lung transplantation (LTx). The classical and genomic investigation of these isolates revealed a common mucoid phenotype and only subtle differences in the genomes. Isolates originating from an individual patient shared ≥98.7% average nucleotide identity (ANI). However, when considering isolates from different patients, substantial variations in terms of sequence type (ST), virulence factors and antimicrobial resistance (AMR) genes were observed. Whole genome multi-locus sequence typing (MLST) confirmed the presence of unique STs per patient regardless of the time from LTx. It was supported by the monophyletic clustering found in the genome-wide phylogeny. The antibiogram shows that ≥91.6% of the isolates were susceptible to amikacin, colistin and tobramycin. For other antibiotics from the panel, isolates frequently showed resistance. Alternatively, a comparative analysis of the 36 P. aeruginosa isolates with 672 strains isolated from diverse ecologies demonstrated clustering of the CF isolates according to the LTx patients from whom they were isolated. We observed that despite LTx and associated measures, all patients remained persistently colonized with similar isolates. The present study shows how whole genome sequencing (WGS) along with phenotypic analysis can help us understand the evolution of P. aeruginosa over time especially its antibiotic resistance.

Synthesis and Antimicrobial Activity of Phosphonopeptide Derivatives Incorporating Single and Dual Inhibitors.

In diagnostic microbiology, culture media are widely used for detection of pathogenic bacteria. Such media employ various ingredients to optimize detection of specific pathogens such as chromogenic enzyme substrates and selective inhibitors to reduce the presence of commensal bacteria. Despite this, it is rarely possible to inhibit the growth of all commensal bacteria, and thus pathogens can be overgrown and remain undetected. One approach to attempt to remedy this is the use of "suicide substrates" that can target specific bacterial enzymes and selectively inhibit unwanted bacterial species. With the purpose of identifying novel selective inhibitors, six novel phosphonopeptide derivatives based on d/l-fosfalin and ß-chloro-l-alanine were synthesized and tested on 19 different strains of clinically relevant bacteria. Several compounds show potential as useful selective agents that could be exploited in the recovery of several bacterial pathogens including Salmonella, Pseudomonas aeruginosa, and Listeria.

C-Terminal 1-Aminoethyltetrazole-Containing Oligopeptides as Novel Alanine Racemase Inhibitors.

In clinical culture media inoculated with patient samples, selective inhibition of commensal bacteria is essential for accurate diagnosis and effective treatment, as they can mask the presence of pathogenic bacteria. The alanine analogue, 1-aminoethyltetrazole was investigated as a potential alanine racemase inhibitor. For effective uptake and enhanced and selective antibacterial activity, a library of C-terminal 1-aminoethyltetrazole containing di- and oligopeptides were synthesized by solid phase peptide coupling techniques. The investigation of the antimicrobial activity of the synthesised compounds identified several clinically applicable selective inhibitors. These enabled differentiation between the closely related bacteria, Salmonella and Escherichia coli, which can be difficult to discriminate between in a clinical setting. In addition, differentiation between enterococci and other Gram-positive cocci was also seen.

Differences and overlaps between Phd studies in diagnostic microbiology in industrial and academic settings.

Industrial and academic needs for innovation and fundamental research are essential and not widely different. Depending on the industrial setting, research and development (R&D) activities may be more focused on the developmental aspects given the need to ultimately sell useful products. However, one of the biggest differences between academic and industrial R&D will usually be the funding model applied and the priority setting between innovative research and product development. Generalizing, companies usually opt for development using customer- and consumer-derived funds whereas university research is driven by open innovation, mostly funded by taxpayer's money. Obviously, both approaches require scientific rigor and quality, dedication and perseverance and obtaining a PhD degree can be achieved in both settings. The formal differences between the two settings need to be realized and students should make an educated choice prior to the start of PhD-level research activities. Intrinsic differences in scientific approaches between the two categories of employers are not often discussed in great detail. We will here document our experience in this field and provide insights into the need for purely fundamental research, industrial R&D and current mixed models at the level of European funding of research. The field of diagnostics in clinical bacteriology and infectious diseases will serve as a source of reference.

Fluorogenic 7-azidocoumarin and 3/4-azidophthalimide derivatives as indicators of reductase activity in microorganisms.

A series of fluorogenic heterocyclic azides were prepared and assessed as reductase substrates across a selection of Gram-negative and Gram-positive microorganisms. The majority of these azides showed similar activity profiles to nitroreductase substrates. Microorganisms that do not produce hydrogen sulfide reduced the azides, indicating reductase activity was not linked to hydrogen sulfide production.

Fluorogenic l-alanylaminopeptidase substrates derived from 6-amino-2-hetarylquinolines and 7-amino-3-hetarylcoumarins and their potential applications in diagnostic microbiology.

Six novel fluorogenic enzyme substrates for detecting l-alanylaminopeptidase activity in microorganisms have been prepared and evaluated in Columbia agar media. The substrates are l-alanyl derivatives of 6-amino-2-hetarylquinolines and 7-amino-3-hetarylcoumarins. Both the quinoline and coumarin series of substrates produced fluorescence in the presence of Gram-negative microorganisms. In contrast, fluorescence generation in the presence of the Gram-positive microorganisms and yeasts was limited or absent.

Strong increase in the autofluorescence of cells signals struggle for survival.

Prokaryotic and eukaryotic cells exhibit an intrinsic natural fluorescence due to the presence of fluorescent cellular structural components and metabolites. Therefore, cellular autofluorescence (AF) is expected to vary with the metabolic states of cells. We examined how exposure to the different stressors changes the AF of Escherichia coli cells. We observed that bactericidal treatments increased green cellular AF, and that de novo protein synthesis was required for the observed AF increase. Excitation and emission spectra and increased expression of the genes from the flavin biosynthesis pathway, strongly suggested that flavins are major contributors to the increased AF. An increased expression of genes encoding diverse flavoproteins which are involved in energy production and ROS detoxification, indicates a cellular strategy to cope with severe stresses. An observed increase in AF under stress is an evolutionary conserved phenomenon as it occurs not only in cells from different bacterial species, but also in yeast and human cells.

Methods for the detection and identification of pathogenic bacteria: past, present, and future.

In order to retard the rate of development of antibacterial resistance, the causative agent must be identified as rapidly as possible, so that directed patient treatment and/or contact precautions can be initiated. This review highlights the challenges associated with the detection and identification of pathogenic bacteria, by providing an introduction to the techniques currently used, as well as newer techniques that are in development. Focusing on the chemical basis for these techniques, the review also provides a comparison of their advantages and disadvantages.

Identification of Enterococcus faecalis enzymes with azoreductases and/or nitroreductase activity.

BACKGROUND: Nitroreductases, NAD(P)H dependent flavoenzymes, are found in most of bacterial species. Even if Enterococcus faecalis strains seems to present such activity because of their sensitivity to nitrofurans, no enzyme has been described. Nitroreductases were separated of others reductases due to their capacity to reduce nitro compounds. They are further classified based on their preference in cofactor: NADH and/or NADPH. However, recently, azoreductases have been studied for their strong activity on nitro compounds, especially nitro pro-drugs. This result suggests a crossing in azo and nitro reductase activities. For the moment, no nitroreductase was demonstrated to possess azoreductase activity. But due to sequence divergence and activity specificity linked to substrates, activity prediction is not evident and biochemical characterisation remains necessary. Identifying enzymes active on these two classes of compounds: azo and nitro is of interest to consider a common physiological role. RESULTS: Four putative nitroreductases, EF0404, EF0648, EF0655 and EF1181 from Enterococcus faecalis V583 were overexpressed as his-tagged recombinant proteins in Escherichia coli and purified following a native or a denaturing/renaturing protocol. EF0648, EF0655 and EF1181 showed nitroreductase activity and their cofactor preferences were in agreement with their protein sequence phylogeny. EF0404 showed both nitroreductase and azoreductase activity. Interestingly, the biochemical characteristics (substrate and cofactor specificity) of EF0404 resembled the properties of the known azoreductase AzoA. But its sequence matched within nitroreductase group, the same as EF0648. CONCLUSIONS: We here demonstrate nitroreductase activity of the putative reductases identified in the Enterococcus faecalis V583 genome. We identified the first nitroreductase able to reduce directly an azo compound, while its protein sequence is close to others nitroreductases. Consequently, it highlights the difficulty in classifying these enzymes solely on the basis of protein sequence alignment and hereby the necessity to experimentally demonstrate the activity. The results provide additional data to consider a broader functionality of these reductases.

Detection of l-alanylaminopeptidase activity in microorganisms using chromogenic self-immolative enzyme substrates.

Three potential chromogenic enzymatic probes, each possessing a self-immolative spacer unit, were synthesised for the purpose of detecting l-alanylaminopeptidase activity in microorganisms. An Alizarin-based probe was the most effective, allowing several species to generate strongly coloured colonies in the presence of metal ions.

Antibiotic Susceptibility Testing of the Gram-Negative Bacteria Based on Flow Cytometry.

Rapidly treating infections with adequate antibiotics is of major importance. This requires a fast and accurate determination of the antibiotic susceptibility of bacterial pathogens. The most frequently used methods are slow because they are based on the measurement of growth inhibition. Faster methods, such as PCR-based detection of determinants of antibiotic resistance, do not always provide relevant information on susceptibility, particularly that which is not genetically based. Consequently, new methods, such as the detection of changes in bacterial physiology caused by antibiotics using flow cytometry and fluorescent viability markers, are being explored. In this study, we assessed whether Alexa Fluor® 633 Hydrazide (AFH), which targets carbonyl groups, can be used for antibiotic susceptibility testing. Carbonylation of cellular macromolecules, which increases in antibiotic-treated cells, is a particularly appropriate to assess for this purpose because it is irreversible. We tested the susceptibility of clinical isolates of Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, to antibiotics from the three classes: ß-lactams, aminoglycosides, and fluoroquinolones. In addition to AFH, we used TO-PRO®-3, which enters cells with damaged membranes and binds to DNA, and DiBAC4 (3), which enters cells with depolarized membranes. We also monitored antibiotic-induced morphological alterations of bacterial cells by analyzing light scattering signals. Although all tested dyes and light scattering signals allowed for the detection of antibiotic-sensitive cells, AFH proved to be the most suitable for the fast and reliable detection of antibiotic susceptibility.

The Synthesis of L-Alanyl and ß-Alanyl Derivatives of 2-Aminoacridone and Their Application in the Detection of Clinically-Important Microorganisms.

In clinical microbiology the speed with which pathogenic microorganisms may be detected has a direct impact on patient health. One important strategy used in the laboratory is the growth of cultures in the presence of an enzymatic substrate which, once transformed by the appropriate microbial enzyme, generates a detectable colour or fluorescence output. Such substrates have previously been prepared by our group and others and are available as commercial diagnostic kits, however they all suffer from some degree of diffusion when used in a solid growth medium. This diffusion complicates the detection and differentiation of species in polymicrobial cultures and so we sought to improve on our previous work. In this work we have prepared and evaluated a series of novel fluorogenic enzyme substrates based on N-substituted-2-aminoacridones. All of the prepared substrates were found to be suitable for the detection and differentiation of certain microorganisms, however those based on the 2-amino-10-benzylacridone core in particular showed no apparent diffusion when incorporated into solid growth media. On transformation these substrates generated brightly fluorescent colonies that are clearly contrasted with the background medium due to the difference in emission wavelength (λem 445-450 nm for the substrate, λem 550 nm for the product). Here we have shown that our L-alanyl aminopeptidase substrate, 2-(N-L-alanylamino)-10-benzylacridone, is particularly suited to the detection of Gram-negative bacteria, and our ß-alanyl aminopeptidase substrate, 2-(N- ß-alanylamino)-10-benzylacridone, to the detection of Pseudomonas aeruginosa and Serratia marcescens when grown on solid media incorporating these substrates. The resulting fluorophore shows no apparent diffusion from the colonies of interest, and the enhanced sensitivity offered by fluorescent emission may allow for the detection of these organisms as microcolonies using automated fluorescence microscopy.

Detection of l-alanylaminopeptidase activity in microorganisms using fluorogenic self-immolative enzyme substrates.

A series of fluorogenic enzymatic substrates that incorporate a self-immolative spacer were synthesised for the purpose of identifying l-alanylaminopeptidase activity in microorganisms in agar media. These substrates resulted in the generation of fluorescent microorganism colonies with Gram-negative microorganisms.

Synthesis and Evaluation of Novel 7- and 8-Aminophenoxazinones for the Detection of ß-Alanine Aminopeptidase Activity and the Reliable Identification of Pseudomonas aeruginosa in Clinical Samples.

A series of novel 8-aminophenoxazin-3-one and 7-aminophenoxazin-3-one chromogens and their corresponding ß-alanine derivatives were synthesized and evaluated for their ability to detect ß-alanyl aminopeptidase activity in bacteria known to hydrolyze ß-alanine derivatized substrates. The results provided insight into the structural requirements for effective visualization of enzymatic activity and the mechanism of formation of phenoxazinon-3-ones. 8-Aminophenoxazin-3-one substrates 23c, 23d, and 23e were prepared in good to high overall yield and were selective for ß-alanyl aminopeptidase activity in bacteria, producing a lighter agar background coloration facilitating visualization of colored colonies, with variable localization to the colonies, but had lower sensitivities for the detection of Pseudomonas aeruginosa in comparison to the analogous 7-aminophenoxazin-3-one substrates. The synthetic methodology employed here allows the preparation of a range of substrates for evaluation and the establishment of structure-activity relationships. For example, the 2-pentyl substituted aminophenoxazin-3-one 22b performed with analogous sensitivity to the corresponding 1-pentyl-7-aminophenoxazin-3-one substrate 1 used commercially, highlighting that the position of the pentyl substituent can be varied while maintaining detection sensitivity.