Enterobacteriaceae detection

Nucleotidylation - the addition of adenylate-residues by Lnu enzymes - can also be the cause of resistance to lincosamide antibiotics in staphylococci and enterococci. A plasmid encoded ADP-ribosylating transferase (Arr-2) that leads to rifampicin resistance has been detected in various Enterobacteriaceae as well as in Pseudomonas aeruginosa. 

Levasseur, S. Husson, M. O. Leitz, R. Merlin, F. Laurent, F. Peladan, F. Drocourt, J. L. Leclerc, H. Van Hoegaerden, M. Rapid detection of members of the family Enterobacteriaceae by a monoclonal antibody. Appl. Environ. Microbiol. 1992,58,1524-1529. 

Mittelman, M. W. Habash, M. Lacroix, J. M. Khoury, A. E. Krajden, M. Rapid detection of Enterobacteriaceae in urine by fluorescent 16S rRNA in situ hybridization on membrane filters. J. Microbiol. Meth. 1997, 30,153-160. 

Enterobacteriaceae sample of 100 ml shall be filtrated and placed the membrane filter into 50 ml of MacConkey broth, incubated for 20 to 28 hours at 35 2°C. Transfer into EMB agar for 40 to 48 hours at 35 2°C. If any characteristic colony is detected, identify using API and/or automated identification system... 

For nonsterile area (class 100,000), the detect count for ten fingers is not more than 50 CFUs/gloves, absence of enterobacteriaceae. 

The enumeration of Enterobacteriaceae and Escherichia coli in water using SPC has been the subject of several studies. These microorganisms serve as indicators of faecal contamination as part of the monitoring of the quality of raw and partially purified waters. They are also used to demonstrate the compliance of a final product with legal standards. For the selective detection of viable Enterobacteriaceae, Baudart et al. (2002,2005) used a nucleic acid probe targetting the 16S rRNA. In order to enumerate the viable cells and to increase the fluorescence intensity, FISH was combined with a DVC procedure and TSA. Using this approach, as little as one fluorescent target cell could be demonstrated in the presence of lO -lO non-fluorescent other cells. 

Aurell H, Catala P, Farge P, WaUet F, Le Brun M, Helbig JH, Jarraud S, Lebaron P (2004) Rapid detection and enumeration of Legionella pneumophila in hot water systems by soUd-phase cytometry. Appl Environ Microbiol 70 1651-1657 Baudart 1, CoaUier 1, Laurent P, Prevost M (2002) Rapid and sensitive enumeration of viable diluted cells of members of the family Enterobacteriaceae in freshwater and drinking water. Appl Environ Microbiol 68 5057-5063... 

In another study (114), three different immobilization methods were evaluated for the detection of E. coli using an antibody coated crystal. The best results were obtained with a protein A coated method, and a response for 10 - lO cells 1 ml of E. coli K12 and other antigens of the Enterobacteriaceae family was observed. Repeated usage of the coated crystal by removing the bound antigen with urea or glycine HCl buffer (pH 2.6) was not very successful. 

Like E. coli, Salmonella spp. is a member of the Enterobacteriaceae family often isolated from humans and food products. The various types of Salmonella spp are known pathogens. S. typhi causes typhoid fever while other species cause diarrhea or even septicemia. Its detection and identification therefore is important for food safety. Common food products that harbor Salmonella spp include eggs or poultry products that have not been properly processed or cooked. 

Table 8.3 Conventional and rapid detection and characterisation methods for heer spoilage acetic acid bacteria, Zymomonas, and Enterobacteriaceae species...

The application of MS-based enzyme activity to detect antibiotic-resistant bacteria was first reported by Hrab et al. where carbapenemase activity was detected using MALDl-MS in viable intact cells of Enterobacteriaceae and Pseudomonas spp. (Hrabak et al. 2011) and later expanded to Acinetobacter baumannii (Hrab et al. 2012). Carbapenemase activity detection in bacteria with MALDl-MS re-qtrires the incubation of viable bacteria (in a suitable buffer system, e.g., 20 mM Tris-HCl, 0.01% sodirrm dodecyl srrlfate (SDS), pH 7.0) with the substrate mol-ecttle, in this case, the antibiotic molectrle (e g., meropenem). This snspension is incnbated for 2 h at 35 °C, in which period meropenem molecules are enzymati-... 

Carbapenemase-producing Enterobacteriaceae (CPE) have already been detected all over the globe with a marked endemicity according to the enzyme type. In Europe, the most critical situation has been reported in Greece and Italy with 60.5 and 28.8% incidence, respectively, of caibapenem-resistant K. pneumoniae isolates recovered from blood samples within the EARS-Net project in 2012 (ECDC 2012). Infections caused by CPE are connected with a significant mortality. Mortality rates observed in small chnical studies ranged from 22 to 72% (Hirsch and Tam 2010 ... 

Since 2011, two new direct assays for detection of carbapenemases have been developed (MALDI-TOF MS hydrolysis assay and Carba NP). Both are designed to substitute reference spectrophotometric assays. Contrary to the spectrophotometric assay and the Carba NP test, the MALDI-TOF MS hydrolysis assay allows for direct detection of hydrolysis products, which imderpins the specificity of the test. By February 2015, three studies focused on a comparison of the MALDI-TOF MS hydrolysis assay with Carba NP (Knox et al. 2014 Papagiannitsis et al. 2015 Chong et al. 2015). Contrary to Knox et al. who foimd similar sensitivity for both tests, two others found better sensitivity in the MALDI-TOF MS hydrolysis assay in Enterobacteriaceae. As already mentioned, the MALDI-TOF MS hydrolysis assay can give excellent results for the detection of CHDL in Acinetobacter baumannii (Kempf et al. 2012). 

These recent data indicate that MALDI-TOF MS has the potential to directly detect the most clinically important AmpC P-lactamases, such as the CMY-2-like, ACC, and DHA types, in clinical isolates of Enterobacteriaceae. In agreement with other MALDI-TOF MS applications (Hrabak 2013), the described protocol is quick and economical. In addition, detection of p-lactamases by MALDI-TOF MS in a proteomic approach allowing the study of the behavior of the tested strains can complement the already used techniques for characterization of P-lactamases, such as PCR and isoelectric focusing (lEF). MALDI-TOF MS can directly detect the class A (Camara and Hays 2007) and class C p-lactamases, as well as other mechanisms such as methylation of rRNA and cell wall components (Cai et al. 2012 Hrabak et al. 2013). We conclude that establishing a MALDI-TOF supplementary database of resistance mechanisms would promote further research in this field. 

Chong PM, McCorrister SJ, Unger MS, Boyd DA, Mulvey MR, Westmacott GR. MALDI-TOF MS detection of carbapenemase activity in clinical isolates of Enterobacteriaceae spp.. Pseudomonas aeruginosa, and Acinetobacter baumannii compared against the Carba-NP assay. J Microbiol Methods. 2015 11 21-3. 

Knox J, Jadhav S, Sevior D, Agyekum A, Whipp M, Waring L, Iredell J, Palombo E. Phenotypic detection of carbapenemase-producing Enterobacteriaceae by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry and the Carba NP test. J Clin Microbiol. 2014 52 4075 7. 

Studentova V, Papagiannitsis CC, Izdebski R, Pfeifer Y, Chudackova E, Bergerova T, Gniadkows-ki M, Hrabak J. Detection of OXA-48-type carbapenemase-producing Enterobacteriaceae in diagnostic laboratories can be enhanced by addition of bicarbonates to cultivation media or reaction buffers. Folia Microbiol. 2015 60 119-29. 

Recently, the molecular basis of cellulose biosynthesis has been detected in S. typhimurium and other Enterobacteriaceae. With this discovery, however, new questions did arise concerning various aspects such as the mode of cellulose biosynthesis, its regulation, function, epidemiology, structure and interaction of cellulose with other components. At present, answers are only partially available, if at all. The availability of well characterized and fully sequenced strains together with efficient tools for genetic manipulation, however, gives hope that fairly soon light will be shed at least to some aspects of cellulose biosynthesis in Enterobacteriaceae. 

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