Sylvain Orenga

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.

Enzymatic substrates in microbiology.

Enzymatic substrates are powerful tools in biochemistry. They are widely used in microbiology to study metabolic pathways, to monitor metabolism and to detect, enumerate and identify microorganisms. Synthetic enzymatic substrates have been customized for various microbial assays, to detect an expanding range of both new enzymatic activities and target microorganisms. Recent developments in synthetic enzymatic substrates with new spectral, chemical and biochemical properties allow improved detection, enumeration and identification of food-borne microorganisms, clinical pathogens and multi-resistant bacteria in various sample types. In the past 20 years, the range of synthetic enzymatic substrates used in microbiology has been markedly extended supporting the development of new multi-test systems (e.g., Microscan, Vitek 2, Phoenix) and chromogenic culture media. The use of such substrates enables an improvement in time to detection and specificity over conventional tests that employ natural substrates. In the era of intense developments in molecular biology, phenotypic tests involving enzymatic substrates remain useful to analyse both simple and complex samples. Such tests are applicable to diagnostic and research laboratories all over the world.

Evaluation of a Chromogenic Culture Medium for Isolation of Clostridium difficile within 24 Hours

ABSTRACT Rapid and effective methods for the isolation of Clostridium difficile from stool samples are desirable to obtain isolates for typing or to facilitate accurate diagnosis of C. difficile-associated diarrhea. We report on the evaluation of a prototype chromogenic medium (ID C. difficile prototype [IDCd]) for isolation of C. difficile. The chromogenic medium was compared using (i) 368 untreated stool samples that were also inoculated onto CLO medium, (ii) 339 stool samples that were subjected to alcohol shock and also inoculated onto five distinct selective agars, and (iii) standardized suspensions of 10 C. difficile ribotypes (untreated and alcohol treated) that were also inoculated onto five distinct selective agars. Two hundred thirty-six isolates of C. difficile were recovered from 368 untreated stool samples, and all but 1 of these strains (99.6%) were recovered on IDCd within 24 h, whereas 74.6% of isolates were recovered on CLO medium after 48 h. Of 339 alcohol-treated stool samples cultured onto IDCd and five other selective agars, C. difficile was recovered from 218 samples using a combination of all media. The use of IDCd allowed recovery of 96.3% of isolates within 24 h, whereas 51 to 83% of isolates were recovered within 24 h using the five other media. Finally, when they were challenged with pure cultures, all 10 ribotypes of C. difficile generated higher colony counts on IDCd irrespective of alcohol pretreatment or duration of incubation. We conclude that IDCd is an effective medium for isolation of C. difficile from stool samples within 24 h.

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 β-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.

A novel chromogenic medium for isolation of Pseudomonas aeruginosa from the sputa of cystic fibrosis patients

BACKGROUND A novel chromogenic medium for isolation and identification of Pseudomonas aeruginosa from sputa of cystic fibrosis (CF) patients was evaluated and compared with standard laboratory methods. METHODS One hundred sputum samples from distinct CF patients were cultured onto blood agar (BA), Pseudomonas CN selective agar (CN) and a Pseudomonas chromogenic medium (PS-ID). All Gram-negative morphological variants from each medium were subjected to antimicrobial susceptibility testing, and identification using a combination of biochemical and molecular methods. RESULTS P. aeruginosa was isolated from 62 samples after 72 h incubation. Blood agar recovered P. aeruginosa from 56 samples (90.3%) compared with 59 samples (95.2%) using either CN or PS-ID. The positive predictive value of PS-ID (98.3%) was significantly higher than growth on CN (88.5%) for identification of P. aeruginosa (P<0.05). CONCLUSIONS PS-ID is a promising medium allowing for the isolation and simultaneous identification of P. aeruginosa from sputa of CF patients.

Characteristics of major Escherichia coli reductases involved in aerobic nitro and azo reduction.

Escherichia coli is able to reduce azo compounds such as methyl red (MR) and nitro compounds such as 7‐nitrocoumarin‐3‐carboxylic acid (7NCCA). The aim of this study was to clarify the specificity of the major E. coli reductases.

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-(β-alanyl)amino-5,7-dialkylnaphthyridine substrates were selectively hydrolysed by β-alanylaminopeptidase expressing bacteria, but were subject to diffusion in the agar medium. Diffusion was reduced in the 2-N-(β-alanyl)amino-7-alkylnaphthyridine substrates with longer alkyl chains, but inhibition of growth was increased. 2-N-(β-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.

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: β-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.

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 β-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 β-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 β-galactosidase activity and generated strongly fluorescent colonies with coliform bacteria that produced this enzyme (e.g., Escherichia coli).