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.

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.

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.

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.

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.

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.

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.

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.

2-(Nitroaryl)benzothiazole and benzoxazole derivatives as fluorogenic substrates for the detection of nitroreductase activity in clinically important microorganisms.

A series of carboxy-substituted 2-(nitroaryl)benzothiazole derivatives and carboxy-substituted 2-(nitroaryl)benzoxazole derivatives were prepared and evaluated as potential nitroreductase substrates for the purpose of detecting clinically important microorganisms. Several of the substrates produced highly fluorescent colonies with the majority of a panel of 10 Gram-negative bacteria and also with two of a panel of 8 Gram-positive bacteria.

Synthesis of 2-arylbenzothiazole derivatives and their application in bacterial detection.

A series of 2-arylbenzothiazole derivatives have been prepared as fluorogenic enzyme substrates in order to detect aminopeptidase, esterase, phosphatase and ß-galactosidase activity in clinically important Gram-negative and Gram-positive bacteria. Substrates were incorporated into an agar-based culture medium and this allowed growth of intensely fluorescent bacterial colonies based on hydrolysis by specific enzymes. Substrate 20 targeted L-alanine aminopeptidase activity and was hydrolysed exclusively by a range of Gram-negative bacteria and inhibited the growth of a range of Gram-positive bacteria. Substrate 19a targeted ß-alanyl aminopeptidase activity and generated fluorescent colonies of selected Gram-negative species including Pseudomonas aeruginosa. Substrate 21b targeted C8-esterase activity and resulted in strongly fluorescent colonies of selected species known to harbour such enzyme activity (e.g., Salmonella and Pseudomonas). Most Gram-negative species produced colonies with an intense blue fluorescence due to hydrolysis of phosphatase substrates 24a-c and substrate 24c was also hydrolysed by strains of Staphylococcus aureus. Compounds 26b and 26c targeted ß-galactosidase activity and generated strongly fluorescent colonies with coliform bacteria that produced this enzyme (e.g., Escherichia coli).

Novel chromogenic aminopeptidase substrates for the detection and identification of clinically important microorganisms.

A series of amino acid derivatives 8-10, 42 and 43 have been prepared as chromogenic enzyme substrates in order to detect aminopeptidase activity in clinically important Gram-negative and Gram-positive bacteria. Enzymatic hydrolysis liberates the amino acid moiety and either a 4-aminophenol or a 4-dialkylaminoaniline derivative which undergoes oxidative coupling with 1-naphthol or a substituted 1-naphthol giving an indophenol dye. Substrates and 1-naphthols were incorporated into an agar-based culture medium and this allowed growth of intensely coloured bacterial colonies based on hydrolysis by specific enzymes. Red/pink coloured colonies were produced by the substrates 8-10 and blue coloured colonies were formed by the substrates 42 and 43. The L-alanyl aminopeptidase substrates 8 targeted L-alanyl aminopeptidase activity and gave coloured colonies with a range of Gram-negative bacteria. Substrates 9 targeted ß-alanyl aminopeptidase activity and generated coloured colonies with selected Gram-negative species including Pseudomonas aeruginosa. Three substrates for L-pyroglutamyl acid aminopeptidase (10a, 10c and 43) were hydrolysed by enterococci and Streptococcus pyogenes to generate coloured colonies. Two yeasts were also included in the study, but they did not produce coloured colonies with any of the substrates examined.

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.

Synthesis and evaluation of fluorogenic 2-amino-1,8-naphthyridine derivatives for the detection of bacteria.

Several novel fluorogenic N-aminoacylnaphthyridine substrates were synthesized in good yield and tested for their ability to detect pathogenic bacteria in agar-based cell culture. Simple 2-N-(ß-alanyl)amino-5,7-dialkylnaphthyridine substrates were selectively hydrolysed by ß-alanylaminopeptidase expressing bacteria, but were subject to diffusion in the agar medium. Diffusion was reduced in the 2-N-(ß-alanyl)amino-7-alkylnaphthyridine substrates with longer alkyl chains, but inhibition of growth was increased. 2-N-(ß-Alanyl)amino-7-octylnaphthyridine inhibited the growth of all species tested, except for strains resistant to colistin/polymyxin, providing a rationale for the development of substrates for the selective detection of drug resistant species in clinical samples.

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.

Prevalence of faecal carriage of Enterobacteriaceae with NDM-1 carbapenemase at military hospitals in Pakistan, and evaluation of two chromogenic media.

OBJECTIVES: To determine the prevalence and antimicrobial susceptibility of carbapenemase-producing Enterobacteriaceae among hospitalized patients and outpatients attending two military hospitals in Rawalpindi, Pakistan, and to compare the performance of two chromogenic culture media for the isolation of these organisms. METHODS: Stool samples from 200 distinct patients were cultured on MacConkey agar and subsequently on two chromogenic media-Colorex KPC and a prototype chromogenic medium, ID Carba-designed for the isolation of carbapenemase-producing Enterobacteriaceae. All Gram-negative isolates growing on either chromogenic medium were investigated for carbapenemases by phenotypic and molecular methods. Producers were subjected to susceptibility testing with 40 antimicrobials by VITEK 2 or agar dilution. RESULTS: In total, 64 NDM-1-positive isolates of Enterobacteriaceae, belonging to seven distinct species, were recovered from 37 (18.5%) of the stool samples. No other carbapenemase types were confirmed. Nineteen positive samples were identified among 70 from inpatients (prevalence 27.1%) and there were 18 positive samples among 130 from outpatients (prevalence 13.8%). Fifty-six isolates (87.5%) harbouring the NDM-1 enzyme were recovered on ID Carba compared with 41 isolates (64.1%) on Colorex KPC (P = 0.012). Multidrug resistance was prevalent, but no pan-resistant isolates were found, with most isolates susceptible in vitro to colistin (97%), mecillinam (95%), fosfomycin (94%), tigecycline (89%) and nitrofurantoin (78%). CONCLUSIONS: This study shows a high prevalence of multidrug-resistant Enterobacteriaceae with the NDM-1 enzyme in Rawalpindi. The new chromogenic medium, ID Carba, was more sensitive than Colorex KPC and has potential as a screening medium for isolation of Enterobacteriaceae harbouring the NDM-1 enzyme.

2-Arylbenzothiazole, benzoxazole and benzimidazole derivatives as fluorogenic substrates for the detection of nitroreductase and aminopeptidase activity in clinically important bacteria.

A series of 2-(2-nitrophenyl)benzothiazole 7, 2-(2-nitrophenyl)benzoxazole 10 and 2-(2-nitrophenyl)benzimidazole 13 derivatives have been synthesised and assessed as indicators of nitroreductase activity across a range of clinically important Gram negative and Gram positive bacteria. The majority of Gram negative bacteria produced strongly fluorescent colonies with substrates 7 and 10 whereas fluorescence production in Gram positive bacteria was less widespread. The l-alanine 16 and 19 and ß-alanine 21 and 23 derivatives have been prepared from 2-(2-aminophenyl)benzothiazole 14 and 2-(2-aminophenyl)benzoxazole 17. These four compounds have been evaluated as indicators of aminopeptidase activity. The growth of Gram positive bacteria was generally inhibited by these substrates but fluorescent colonies were produced with the majority of Gram negative bacteria tested.