Metabolic enzyme Suclg2 maintains tolerogenicity of regulatory dendritic cells diffDCs by suppressing Lactb succinylation.

Metabolic reprogramming plays a pivotal role in the differentiation and function of immune cells including dendritic cells (DCs). Regulatory DCs can be generated in regional tissue niches like splenic stroma and act as an important part of stromal control of immune response for the maintenance of immune tolerance. However, the metabolic alterations during splenic stroma-driven regulatory DCs differentiation and the metabolic enzyme involved in regulatory DCs function remain poorly understood. By combining metabolomic, transcriptomic, and functional investigations of mature DCs (maDCs) and diffDCs (regulatory DCs differentiated from activated mature DCs through coculturing with splenic stroma), here we identified succinate-CoA ligase subunit beta Suclg2 as a key metabolic enzyme that reprograms the proinflammatory status of mature DCs into a tolerogenic phenotype via preventing NF-κB signaling activation. diffDCs downregulate succinic acid levels and increase the Suclg2 expression along with their differentiation from mature DCs. Suclg2-interference impaired the tolerogenic function of diffDCs in inducing T cell apoptosis and enhanced activation of NF-κB signaling and expression of inflammatory genes CD40, Ccl5, and Il12b in diffDCs. Furthermore, we identified Lactb as a new positive regulator of NF-κB signaling in diffDCs whose succinylation at the lysine 288 residue was inhibited by Suclg2. Our study reveals that the metabolic enzyme Suclg2 is required to maintain the immunoregulatory function of diffDCs, adding mechanistic insights into the metabolic regulation of DC-based immunity and tolerance.

Bacteremia with ESBL-producing Enterobacterales is associated with IgG antibodies reacting with CTX-M-15 and/or CTX-M-27.

OBJECTIVES: The adaptive humoral immune response following clinical infection with extended spectrum beta-lactamase (ESBL)-producing Enterobacterales (EPE) has not been thoroughly investigated. The aim of this study was to investigate the presence of anti-CTX-M-15 and/or anti-CTX-M-27 IgG antibodies in bacteremia patients diagnosed with EPE compared to a control group consisting of patients suffering from bacteremia with third generation cephalosporin-susceptible Escherichia coli (3GCSE). METHODS: Patientswith EPE (n = 59) or 3GCSE (n = 42) bacteremia were recruited in this case control study in the Skåne County (South of Sweden). Sera were collected 1-26 months after bacteremia. Enzyme-linked immunosorbent assay (ELISA) was used for detection of specific IgG antibodies directed against recombinant beta-lactamases CTX-M-15 and CTX-M-27. The beta-lactamase resistance genes of the corresponding EPE blood isolates were determined by DNA sequencing. RESULTS: The majority (n = 47; 80 %) of the 59 EPE blood isolates carried blaCTX-M-15 or blaCTX-M-27 genes. IgG antibodies reacting to the corresponding CTX-M enzyme was seen in 28 % (13/47) of patients suffering from EPE-bacteremia, while antibodies were detected in only 9.5 % (4/42) of patients with 3GCSE (p = 0.03). Patients with EPE had a statistically significantly higher median Charlson comorbidity index and prevalence of renal disease (p = 0.01), compared to the 3GCSE control group. CONCLUSION: This study implies that EPE bacteremia can trigger production of IgG antibodies targeting ESBL. Further investigations are required to determine the functional role of anti-ESBL antibodies against EPE bacteremia.

Inhibitory Potential of Polyclonal Camel Antibodies against New Delhi Metallo-ß-lactamase-1 (NDM-1).

New Delhi Metallo-ß-lactamase-1 (NDM-1) is the most prevalent type of metallo-ß-lactamase, able to hydrolyze almost all antibiotics of the ß-lactam group, leading to multidrug-resistant bacteria. To date, there are no clinically relevant inhibitors to fight NDM-1. The use of dromedary polyclonal antibody inhibitors against NDM-1 represents a promising new class of molecules with inhibitory activity. In the current study, immunoreactivities of dromedary Immunoglobulin G (IgG) isotypes containing heavy-chain and conventional antibodies were tested after successful immunization of dromedary using increasing amounts of the recombinant NDM-1 enzyme. Inhibition kinetic assays, performed using a spectrophotometric method with nitrocefin as a reporter substrate, demonstrated that IgG1, IgG2, and IgG3 were able to inhibit not only the hydrolytic activity of NDM-1 but also Verona integron-encoded metallo-ß-lactamase (VIM-1) (subclass B1) and L1 metallo-ß-lactamase (L1) (subclass B3) with inhibitory concentration (IC50) values ranging from 100 to 0.04 µM. Investigations on the ability of IgG subclasses to reduce the growth of recombinant Escherichia coli BL21(DE3)/codon plus cells containing the recombinant plasmid expressing NDM-1, L1, or VIM-1 showed that the addition of IgGs (4 and 8 mg/L) to the cell culture was unable to restore the susceptibility of carbapenems. Interestingly, IgGs were able to interact with NDM-1, L1, and VIM-1 when tested on the periplasm extract of each cultured strain. The inhibitory concentration was in the micromolar range for all ß-lactams tested. A visualization of the 3D structural basis using the three enzyme Protein Data Bank (PDB) files supports preliminarily the recorded inhibition of the three MBLs.

Ongoing dissemination of OXA-244 carbapenemase-producing Escherichia coli in Switzerland and their detection.

OXA-244 is a derivative of OXA-48 showing weaker carbapenemase activity, compromising the detection of corresponding producers in clinical laboratories. Since 2017, the Swiss National Reference Center for Emerging Antibiotic Resistance noticed an increased identification of OXA-244-producing Escherichia coli (n=15) within the country. Different methods (biochemical and immunoassay tests, screening culture media) were tested for the detection of OXA-244 producers. Whole genome sequencing was used to investigate the genetic relatedness between the isolates and the genetic structures at the origin of the acquisition of the blaOXA-244 gene. The mSuperCARBA® medium and the NG-Test CARBA5 assay were found to be suitable tools for detecting all OXA-244-producing isolates. Other selective media did not perform optimally. Among the fifteen strains, five sequence types were identified, with ST38 being predominant. The blaOXA-244 gene was located on the chromosome for all isolates. Overall, detection of OXA-244 producers is challenging and specific guidelines must be followed.

Development of a Chimeric Vaccine Against Pseudomonas aeruginosa Based on the Th17-Stimulating Epitopes of PcrV and AmpC.

Pulmonary infection caused by Pseudomonas aeruginosa (PA) has created an urgent need for an efficient vaccine, but the protection induced by current candidates is limited, partially because of the high variability of the PA genome. Antigens targeting pulmonary Th17 responses are able to provide antibody-independent and broad-spectrum protection; however, little information about Th17-stimulating antigens in PA is available. Herein, we identified two novel PA antigens that effectively induce Th17-dependent protection, namely, PcrV (PA1706) and AmpC (PA4110). Compared to intramuscular immunization, intranasal immunization enhanced the protection of rePcrV due to activation of a Th17 response. The Th17-stimulating epitopes of PcrV and AmpC were identified, and the recombinant protein PVAC was designed and generated by combining these Th17-stimulating epitopes. PVAC was successfully produced in soluble form and elicited broad protective immunity against PA. Our results provide an alternative strategy for the development of Th17-based vaccines against PA and other pathogens.

Generation and selection of antibodies for a novel immunochromatographic lateral flow test to rapidly identify OXA-23-like-mediated carbapenem resistance in Acinetobacter baumannii.

INTRODUCTION: The spread of carbapenem-resistant Acinetobacter baumannii has led to a worldwide healthcare problem. Carbapenem resistance in A. baumannii is mainly mediated by the acquisition of the carbapenem-hydrolyzing oxacillinase OXA-23. The phenotypic detection of carbapenem-producing A. baumannii is challenging and time-consuming. Hence, there is an unmet medical need for reliable and rapid diagnostic tools to detect OXA-23-producing Acinetobacter isolates to enable successful patient management. AIM: Development of an immunochromatographic lateral flow test (ICT) for the rapid and reliable detection of OXA-23-producing carbapenem-resistant Acinetobacter isolates. METHODOLOGY: For the development of an antibody-based ICT, we generated anti-OXA-23 monoclonal antibodies (MoAbs) and screened them sequentially for their ability to bind native OXA-23. Selected OXA-23-specific MoAbs were tested in different combinations for their capacity to capture and detect OXA-23His6 by sandwich enzyme-linked immunosorbent assay (ELISA) and ICT. A well-characterized collection of carbapenem-resistant Acinetobacter isolates with defined carbapenem resistance mechanisms were used to evaluate the specificity of the final OXA-23 ICT prototype. RESULTS: The antibody pairs best suited for the sandwich ELISA format did not match the best pairs in the ICT format selected during the development process of the final prototype OXA-23 ICT. This prototype was able to differentiate between OXA-23 subfamily-mediated carbapenem resistance and carbapenem-resistant Acinetobacter isolates overexpressing other OXAs with 100  % specificity and a turnaround time of 20 min from culture plate to result. CONCLUSION: With this rapid detection assay one can save 12-48 h of diagnostic time, which could help avoid inappropriate use of carbapenems and enable earlier intervention to control the transmission of OXA-23-producing carbapenem-resistant Acinetobacter isolates to other patients and healthcare workers.

Assessment of a newly developed immunochromatographic assay for NDM-type metallo-ß-lactamase producing Gram-negative pathogens in Myanmar.

BACKGROUND: To detect carbapenemase-producing Gram-negative bacteria in bacterial laboratories at medical settings, a new immunochromatographic assay for New Delhi metallo-ß-lactamases (NDMs) was developed. METHODS: The immunochromatographic assay for New Delhi metallo-ß-lactamases producers was developed using rat monoclonal antibodies against NDMs. The assessment was performed using 350 isolates of Gram-negative bacteria, including Acinetobacter baumannii (51 isolates), Enterobacteriaceae (163 isolates), and Pseudomonas aeruginosa (136 isolates) obtained from 2015 to 2017 in medical settings in Myanmar. Of them, 302 isolates were resistant to carbapenems, including imipenem and/or meropenem. The blaNDM genes were identified by PCR and sequencing. RESULTS: Of the 350 clinical isolates tested, 164 (46.9%) (60 isolates of Escherichia coli, 51 isolates of Klebsiella pneumoniae, 25 isolates of Enterobacter cloacae, 23 isolates of P. aeruginosa, and 5 isolates of A. baumannii) were positive on this assay, and all the positive isolates harbored genes encoding NDM-1, - 4, - 5 and - 7. The remaining 186 (53.1%) isolates negative on the assay did not harbor genes encoding NDMs. The assay had a specificity of 100% and a sensitivity of 100%. The assessment revealed that more than 90% of carbapenem-resistant Enterobacteriaceae produced NDMs. CONCLUSIONS: The immunochromatographic assay is an easy-to-use and reliable kit for detection of NDMs-producing Gram-negative bacteria. The assay revealed that NDM-producing Enterobacteriaceae isolates are wide-spread in medical settings in Myanmar.

Avibactam Sensitizes Carbapenem-Resistant NDM-1-Producing Klebsiella pneumoniae to Innate Immune Clearance.

Infections caused by New Delhi metallo-ß-lactamase (NDM)-producing strains of multidrug-resistant Klebsiella pneumoniae are a global public health threat lacking reliable therapies. NDM is impervious to all existing ß-lactamase inhibitor (BLI) drugs, including the non-ß-lactam BLI avibactam (AVI). Though lacking direct activity against NDMs, AVI can interact with penicillin-binding protein 2 in a manner that may influence cell wall dynamics. We found that exposure of NDM-1-producing K. pneumoniae to AVI led to striking bactericidal interactions with human cathelicidin antimicrobial peptide LL-37, a frontline component of host innate immunity. Moreover, AVI markedly sensitized NDM-1-producing K. pneumoniae to killing by freshly isolated human neutrophils, platelets, and serum when complement was active. Finally, AVI monotherapy reduced lung counts of NDM-1-producing K. pneumoniae in a murine pulmonary challenge model. AVI sensitizes NDM-1-producing K. pneumoniae to innate immune clearance in ways that are not appreciated by standard antibiotic testing and that merit further study.

Phenotypic Detection of Carbapenemase-Producing Organisms from Clinical Isolates.

The rapid spread of multidrug-resistant Gram-negative organisms constitutes one of the greatest challenges to global health. While Gram-negative organisms have developed several mechanisms to avert the bactericidal effects of commonly prescribed antibiotic agents, the increasing prevalence of carbapenemase-producing organisms (CPO) is particularly concerning given the rapid spread of mobile genetic elements containing carbapenemase genes, the limited treatment options for infections caused by these organisms, and the high mortality rates associated with CPO infections. Understanding if an organism is carbapenemase producing and, if so, the class of carbapenemase(s) produced has treatment implications, as some agents preferentially have activity against specific carbapenemases. Furthermore, CPO disseminate between patients with greater ease than non-CP-carbapenem-resistant organisms and warrant more intensive infection control measures than would be employed in the absence of carbapenemase production. Phenotypic assays currently used in clinical practice to detect CPO consist of the following: (i) growth-based assays which measure carbapenem resistance based on organism growth in the presence of a carbapenem antibiotic (e.g., modified Hodge test and modified carbapenem inactivation method), (ii) hydrolysis methods which detect carbapenem degradation products (e.g., Carba NP test and matrix-assisted laser desorption-ionization time of flight mass spectrometry), and (iii) lateral flow immunoassays which detect carbapenemase enzymes through the use of specific antibodies. Although there is no single phenotypic test that meets all specifications of the ideal test, as we describe in this review, there are a number of tests that are user-friendly, affordable, accurate, and feasible for implementation in clinical microbiology laboratories of all sizes.

A multiplex lateral flow immunoassay for the rapid identification of NDM-, KPC-, IMP- and VIM-type and OXA-48-like carbapenemase-producing Enterobacteriaceae.

Objectives: The global spread of carbapenemase-producing Enterobacteriaceae represents a substantial challenge in clinical practice and rapid and reliable detection of these organisms is essential. The aim of this study was to develop and validate a lateral flow immunoassay (Carba5) for the detection of the five main carbapenemases (KPC-, NDM-, VIM- and IMP-type and OXA-48-like). Methods: Carba5 was retrospectively and prospectively evaluated using 296 enterobacterial isolates from agar culture. An isolated colony was suspended in extraction buffer and then loaded on the manufactured Carba5. Results: All 185 isolates expressing a carbapenemase related to one of the Carba5 targets were correctly and unambiguously detected in <15 min. All other isolates gave negative results except those producing OXA-163 and OXA-405, which are considered low-activity carbapenemases. No cross-reaction was observed with non-targeted carbapenemases, ESBLs, AmpCs or oxacillinases (OXA-1, -2, -9 and -10). Overall, this assay reached 100% sensitivity and 95.3% (retrospectively) to 100% (prospectively) specificity. Conclusions: Carba5 is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for confirmation of the five main carbapenemases encountered in Enterobacteriaceae.

Monoclonal antibody-mediated detection of CTX-M ß-lactamases in Gram-negative bacteria.

Gram-negative bacteria (GNB) that express CTX-M ß-lactamases have become a serious threat to the clinical management of GNB infections. While antibody-based platforms have been successfully used in research settings to study and detect other ß-lactamases-including SHV, CMY, and TEM enzymes-there is currently a lack of antibody-based tools to detect the CTX-M enzymes. Here we describe the development of an anti-CTX-M sandwich ELISA based on a pair of monoclonal antibodies (mAbs)-mAb 6101-33 and mAb 6101-19-used as the capture and detection antibody, respectively. This antibody pair detected CTX-M variants from group 1 (CTX-M-15), group 2 (CTX-M-2), group 8 (CTX-M-8), and group 9 (CTX-M-14) that were expressed by a training set of clinical GNB isolates. The limit of detection for this sandwich ELISA was 30ng of recombinant CTX-M-15, and CTX-Ms expressed by 106 lysed CFU of GNB. When tested against a blinded panel of 78 clinical isolates, the sandwich ELISA demonstrated a sensitivity of 96% and a specificity of 100%. The mAb pair did not cross-react with bacteria that contained other ß-lactamases, including TEM, SHV, OXA, KPC, NDM, CMY, and DHA. In conclusion, we developed a highly sensitive and specific sandwich ELISA, capable of detecting CTX-M enzyme production in GNB pathogens.

Differential Th17 response induced by the two clades of the pandemic ST258 Klebsiella pneumoniae clonal lineages producing KPC-type carbapenemase.

The spread of KPC-type carbapenemases is mainly attributed to the global dissemination of Klebsiella pneumoniae (KP) strains belonging to the clonal group (CG) 258, including sequence type (ST) 258 and other related STs. Two distinct clades of CG258-KP have evolved, which differ mainly for the composition of their capsular polysaccharides, and recent studies indicate that clade 1 evolved from an ancestor of clade 2 by recombination of a genomic fragment carrying the capsular polysaccharide (cps) locus. In this paper, we investigated the ability of two ST258-KP strains, KKBO-1 and KK207-1, selected as representatives of ST258-KP clade 2 and clade 1, respectively, to activate an adaptive immune response using ex vivo-stimulation of PBMC from normal donors as an experimental model. Our data showed that KKBO-1 (clade 2) induces a Th17 response more efficiently than KK207-1 (clade 1): the percentage of CD4+IL17+ cells and the production of IL-17A were significantly higher in cultures with KKBO-1 compared to cultures with KK207-1. While no differences in the rate of bacterial internalization or in the bacteria-induced expression of CD86 and HLA-DR by monocytes and myeloid dendritic cells were revealed, we found that the two strains significantly differ in inducing the production of cytokines involved in the adaptive immune response, as IL-1ß, IL-23 and TNF-α, by antigen-presenting cells, with KKBO-1 being a more efficient inducer than KK207-1. The immune responses elicited by KK207-1 were comparable to those elicited by CIP 52.145, a highly virulent K. pneumoniae reference strain known to escape immune-inflammatory responses. Altogether, present results suggest that CG258-KP of the two clades are capable of inducing a different response of adaptive immunity in the human host.

Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity.

Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 ß-lactamase (P99ßL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide-MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 109 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99ßL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening.

Prospective evaluation of the OKN K-SeT assay, a new multiplex immunochromatographic test for the rapid detection of OXA-48-like, KPC and NDM carbapenemases.

Objectives: There is an urgent need for accurate and fast diagnostic tests capable of identifying carbapenemase producers. Here, we assessed the performance of a new multiplex lateral flow assay (OKN K -SeT) for the rapid detection of OXA-48-like, KPC and NDM carbapenemase-producing Enterobacteriaceae from culture colonies. Methods: Two hundred collection isolates with characterized ß-lactamase content and 183 non-duplicate consecutive isolates referred to two National Reference Centres over a 2 month period in 2016 were used to evaluate the OKN K -SeT assay. Results: The assay correctly detected all 42 OXA-48-like-, 27 KPC- and 30 NDM-producing isolates from the collection panel, including 7 isolates that co-produced NDM and OXA-181 carbapenemases. No cross-reactivity was observed with non-targeted carbapenemases ( n = 41) or with non-carbapenemase producers ( n = 60). Prospectively, all OXA-48-like ( n = 69), KPC ( n = 9) and NDM ( n = 19) carbapenemase-producing Enterobacteriaceae isolates were correctly detected, while 11 carbapenemase producers not targeted by the assay went undetected [VIM ( n = 8) and OXA-23/OXA-58-like ( n = 3)]. Overall, the sensitivity and specificity of the assay were 100%. Conclusions: The OKN assay is efficient, rapid and easy to implement in the workflow of a clinical microbiology laboratory for the confirmation of OXA-48, NDM and KPC carbapenemases. This test represents a powerful diagnostic tool as it enables the rapid detection of the most clinically important carbapenemases without the need for more costly and less frequently available molecular assays.

Multitarget selection of catalytic antibodies with ß-lactamase activity using phage display.

ß-lactamase enzymes responsible for bacterial resistance to antibiotics are among the most important health threats to the human population today. Understanding the increasingly vast structural motifs responsible for the catalytic mechanism of ß-lactamases will help improve the future design of new generation antibiotics and mechanism-based inhibitors of these enzymes. Here we report the construction of a large murine single chain fragment variable (scFv) phage display library of size 2.7 × 109 with extended diversity by combining different mouse models. We have used two molecularly different inhibitors of the R-TEM ß-lactamase as targets for selection of catalytic antibodies with ß-lactamase activity. This novel methodology has led to the isolation of five antibody fragments, which are all capable of hydrolyzing the ß-lactam ring. Structural modeling of the selected scFv has revealed the presence of different motifs in each of the antibody fragments potentially responsible for their catalytic activity. Our results confirm (a) the validity of using our two target inhibitors for the in vitro selection of catalytic antibodies endowed with ß-lactamase activity, and (b) the plasticity of the ß-lactamase active site responsible for the wide resistance of these enzymes to clinically available inhibitors and antibiotics.

Absence of phosphatidylcholine in bacterial membranes facilitates translocation of Sec-dependent ß-lactamase AmpC from cytoplasm to periplasm in two Pseudomonas strains.

Phosphatidylcholine (PC) is a rare membrane lipid in bacteria but crucial for virulence of various plant and animal pathogens. The pcs- mutant lacking PC in bacterial membranes of Pseudomonas syringae pv. syringae van Hall 1336 displayed more ampicillin resistance. Ampicillin susceptibility tests gave an IC50 (half maximal inhibitory concentration) of 52 mg/ml for Pseudomonas syringae pv. syringae van Hall 1336, 53 mg/ml for the complemented strain 1336 RM (pcs-/+) and 90 mg/ml for the 1336 pcs- mutant. Activity assay of ß-lactamase in periplasmic extracts gave 0.050 U/mg for the 1336 wild type, 0.052 U/mg for the 1336RM (pcs-/+), 0.086 U/mg for the 1336 pcs- mutant. Analysis by western blotting showed that the content of AmpC enzyme was markedly different in periplasmic extracts between the wild-type and pcs- mutant strains. Reverse transcriptase PCR also showed that the presence or absence of PC in bacterial membranes did not affect the transcription of ampC gene. The phenotype of the pcs- mutant was able to be recovered to the wild type by introducing a wild-type pcs gene into the pcs- mutant. Similar results were also obtained from the soil-dwelling bacterium Pseudomonas sp. 593. Our results demonstrate that the absence of PC in bacterial membranes facilitates the translocation of Sec-dependent ß-lactamase AmpC from cytoplasm to periplasm, and the enhanced ampicillin-resistance in the pcs- strains mainly comes from effective translocation of AmpC via Sec-pathway.

Colonisation with extended-spectrum beta-lactamase (ESBL) not detected in a prevalence study.

BACKGROUND: The Mid-West of Ireland has higher than average national rates of invasive extended-spectrum beta-lactamase (ESBL) bloodstream infections and carbapenemase-producing Enterobacteriaceae (CPE), with increasing numbers of ESBL isolates detected in community-dwelling patients. AIMS: To conduct a point prevalence study in a convenience sample of the Mid-West population with the aim of determining the extent of ESBL colonisation. METHODS: Utilising anonymised community stool samples that had completed routine analysis, we conducted a point prevalence study over a 4-week period on all samples that met defined inclusion and exclusion criteria. Limited epidemiological data was recorded: (1) age of patient, (2) gender, and (3) sender location. From these stool specimens, rectal swabs were inoculated (eSwab™ 480CE, Copan, Italy), which were subsequently cultured on selective chromogenic agar (Colorex™ ESBL). Culture plates were incubated aerobically at 37 °C for 24 h. RESULTS: Of 195 samples processed, 58 % (n = 112) were from females. The median patient age was 62.4 years (range 20-94 years). 186 samples (95 %) originated from general practitioner clinics. During the study period, only nine eligible stool samples were received from LTCF (6 public). From 195 Colorex™ ESBL chromogenic agar plates cultured, no ESBL-producing organisms were detected. CONCLUSIONS: This community point prevalence study did not identify ESBL colonisation despite high numbers of patients with invasive ESBL bloodstream infections presenting for admission in our institution. We believe this may be because of our small sample size. Data regarding antimicrobial exposure and other risk factors for ESBL colonisation were also not available. We remain vigilant for ESBL-producing organisms.

Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.

Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI.