Listeriosis Caused by Persistence of Listeria monocytogenes Serotype 4b Sequence Type 6 in Cheese Production Environment.

A nationwide outbreak of human listeriosis in Switzerland was traced to persisting environmental contamination of a cheese dairy with Listeria monocytogenes serotype 4b, sequence type 6, cluster type 7488. Whole-genome sequencing was used to match clinical isolates to a cheese sample and to samples from numerous sites within the production environment.

Development of Tyrphostin Analogues to Study Inhibition of the Mycobacterium tuberculosis Pup Proteasome System*.

Tuberculosis is a global health problem caused by infection with the Mycobacterium tuberculosis (Mtb) bacteria. Although antibiotic treatment has dramatically reduced the impact of tuberculosis on the population, the existence and spreading of drug resistant strains urgently demands the development of new drugs that target Mtb in a different manner than currently used antibiotics. The prokaryotic ubiquitin-like protein (Pup) proteasome system is an attractive target for new drug development as it is unique to Mtb and related bacterial genera. Using a Pup-based fluorogenic substrate, we screened for inhibitors of Dop, the Mtb depupylating protease, and identified I-OMe-Tyrphostin AG538 (1) and Tyrphostin AG538 (2). The hits were validated and determined to be fast-reversible, non-ATP competitive inhibitors. We synthesized >25 analogs of 1 and 2 and show that several of the synthesized compounds also inhibit the depupylation actions of Dop on native substrate, FabD-Pup. Importantly, the pupylation activity of PafA, the sole Pup ligase in Mtb, was also inhibited by some of these compounds.

Mycobacterium helveticum sp. nov., a novel slowly growing mycobacterial species associated with granulomatous lesions in adult swine.

The occurrence of nontuberculous mycobacteria in different hosts and their implication as obligate or opportunistic pathogens remain mainly unclear. Mycobacteriosis in pigs is usually associated with members of the Mycobacterium avium complex and, in particular, with 'Mycobacterium avium subsp. hominissuis'. Here we describe a novel slow-growing mycobacterial species isolated from lymph nodes obtained from two sows housed in different Swiss farms. The animals presented chronic inappetence and mild diarrhoea. Gross pathology revealed focal caseous lymphadenopathy of the mesenteric lymph nodes. Complete genome sequencing of the two isolates from the two sows was performed. The genomes comprised 5.76 Mb and an average nucleotide identity score of 99.97 %. Whole genome sequence, mycolic acid and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses revealed that the two isolates were not related to any previously described Mycobacterium species. The closest related species was Mycobacterium parmense, a slow-growing scotochromogenic mycobacterium first isolated from a cervical lymph node of a 3-year-old child. The name proposed for the new species is Mycobacterium helveticum sp. nov. and 16-83T (=DSM 109965T= LMG 2019-02457T) is the type strain.

Incidence, trends and risk factors for perineal injuries of low-risk pregnant women: Experience from a midwife run obstetric unit, South Africa.

Pregnant women experience perineal injuries during childbirth. The objectives of this cross-sectional retrospective study were to estimate the incidence, trends, and risk factors for perineal injuries of women who had childbirths from January 2013 to December 2017. We used logistic regression to identify risk factors for all injuries, episiotomy, and obstetric anal sphincter injury (OASI) measured by odds ratios (OR). A total of 5547 women showed gradual decreases of episiotomy from 17.6% in 2013 to 7.6% in 2017 (p <0.05). Perineal injuries were reduced from 33.3% in 2013 to 28.9% in 2017 (p <0.05). The risk factor for any perineal injury were younger ages, term pregnancy, and nil parity (p,0.05). Advanced gestational age, nil parity, and previous vaginal births were risk factors for episiotomy. However, birth weight of baby was significantly associated with OASI. Episiotomy and overall perineal injury rates were commendable. Training to midwives is needed to improve perineal care and maintain good practices during delivery.

Escape from planarity in fragment-based drug discovery: A physicochemical and 3D property analysis of synthetic 3D fragment libraries.

Fragment-based drug discovery (FBDD) has grown into a well-established approach in the pursuit of new therapeutics. Key to the success of FBDD is the low molecular complexity of the initial hits and this has resulted in fragment libraries that mainly contain compounds with a two-dimensional (2D) shape. In an effort to increase the chemical diversity and explore the impact of increased molecular complexity on the hit rate of fragment library screening, several academic and industrial groups have designed and synthesised novel fragments with a three-dimensional (3D) shape. This review provides an overview of 25 synthetic 3D fragment libraries from the recent literature. We calculate and compare physicochemical properties and descriptors that are typically used to measure molecular three-dimensionality such as fraction sp3 (Fsp3), plane of best fit (PBF) scores and principal moment of inertia (PMI) plots. Although the libraries vary widely in structure and properties, some key common features can be identified which may have utility in designing the next generation of 3D fragment libraries.

First Clinical Case of In Vivo Acquisition of DHA-1 Plasmid-Mediated AmpC in a Salmonella enterica subsp. enterica Isolate.

A pan-susceptible Salmonella enterica serovar Worthington isolate was detected in the stool of a man returning from Sri Lanka. Under ceftriaxone treatment, a third-generation cephalosporin (3GC)-resistant Salmonella Worthington was isolated after 8 days. Molecular analyses indicated that the two isolates were identical. However, the latter strain acquired a blaDHA-1-carrying IncFII plasmid probably from a Citrobacter amalonaticus isolate colonizing the gut. This is the first report of in vivo acquisition of plasmid-mediated resistance to 3GCs in S. enterica.

Acquired Resistance to Bedaquiline and Delamanid in Therapy for Tuberculosis.

Treatment of multidrug-resistant Mycobacterium tuberculosis is a challenge. This letter describes the emergence of resistance to new therapies, bedaquiline and delamanid.

Microwave-assisted diastereoselective two-step three-component synthesis for rapid access to drug-like libraries of substituted 3-amino-ß-lactams.

Large, diverse compound libraries are an essential requisite in target-based drug development. In this work, a robust microwave-assisted synthesis for the diastereoselective generation of 3-saccharinyl-trans-ß-lactams is reported. The method is optimised for combinatorial library synthesis in which decoration of the scaffold is varied on both the ß-lactam and the saccharine moiety. Within the European Lead Factory (ELF) consortium, a library of 263 compounds was efficiently produced using the developed methodology.

Evaluation of the AID carbapenemase line probe assay for rapid detection and identification of carbapenemase genes in Gram-negative bacilli.

Objectives: This study evaluated the AID carbapenemase line probe assay (LPA) for the detection and identification of carbapenem resistance genes in Enterobacteriaceae and other Gram-negative bacilli (GNB) using bacterial cultures and DNA extracts directly from patient urine samples. Methods: The AID carbapenemase LPA detects 13 different carbapenemase genes. Test probe accuracy was verified for using clinical Enterobacteriaceae isolates harbouring bla KPC , bla VIM , bla NDM , bla GIM , bla AIM , bla SPM , bla IMP and bla OXA-48 and a well-characterized set of Escherichia coli DH5α strains transformed with the vector plasmid pUC57- kan harbouring bla BIC , bla SIM , bla DIM , bla IMI-3 , bla IMI-1 and bla NMC-A . Sensitivity and specificity was determined by testing 151 clinical GNB strains previously characterized for the production of carbapenemase activity and carbapenemase genes. Direct detection of carbapenemase genes using the LPA was determined using 299 clinical urine specimens. Analytical sensitivity for detection in urine was determined by testing serial dilutions of bla KPC and bla NDM in clinical Klebsiella pneumoniae strains. Results: All carbapenemase gene probes showed 100% accuracy without cross-reactions. Sensitivity and specificity of the LPA using clinical isolates was 100% for each. Analytical sensitivity for detection of bla KPC and bla NDM in urine was 10 1 -10 2 cfu. The LPA detected carbapenemase genes in 20 urines, which were confirmed in 12 samples by conventional multiplex PCR. Remarkably, 0 of the 20 urines grew carbapenemase-suspicious GNB applying EUCAST recommendations. Conclusions: The AID carbapenemase LPA is an accurate, sensitive and easy-to-use test for the detection and identification of carbapenemase genes, which can readily be implemented in any diagnostic laboratory.

Reemergence of Mycobacterium chimaera in Heater-Cooler Units despite Intensified Cleaning and Disinfection Protocol.

Invasive Mycobacterium chimaera infections after open-heart surgery have been reported internationally. These devastating infections result from aerosols generated by contaminated heater-cooler units used with extracorporeal circulation during surgery. Despite intensified cleaning and disinfection, surveillance samples from factory-new units acquired during 2014 grew nontuberculous mycobacteria after a median of 174 days.

The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis.

Nosocomial infections with Enterococcus faecalis are an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters of E. faecalis that are annotated as drug efflux pumps. Deletion of ef0789-ef0790 on the chromosome of E. faecalis resulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context of E. faecalis In contrast, heterologous expression in Lactococcus lactis revealed that EfrAB, EfrCD, and the product of ef2226-ef2227 (EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression in L. lactis Since all seven transporters were purified as heterodimers after overexpression in L. lactis, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell.

Evaluation of the Bruker MALDI Biotyper for Identification of Fastidious Gram-Negative Rods.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has entered clinical laboratories, facilitating identification of bacteria. Here, we evaluated the MALDI Biotyper (Bruker Daltonics) for the identification of fastidious Gram-negative rods (GNR). Three sample preparation methods, direct colony transfer, direct transfer plus on-target formic acid preparation, and ethanol-formic acid extraction, were analyzed for 151 clinical isolates. Direct colony transfer applied with the manufacturer's interpretation criteria resulted in overall species and genus identification rates of 43.0% and 32.5%, respectively; 23.2% of the isolates were not identified, and two misidentifications (1.3%) were observed. The species identification rates increased to 46.4% and 53.7% for direct transfer plus formic acid preparation and ethanol-formic acid extraction, respectively. In addition, we evaluated score value cutoff alterations. The identification rates hardly increased by reducing the genus cutoff, while reducing the 2.0 species cutoff to 1.9 and to 1.8 increased the identification rates to up to 66.2% without increasing the rate of misidentifications. This study shows that fastidious GNR can reliably be identified using the MALDI Biotyper. However, the identification rates do not reach those of nonfastidious GNR such as the Enterobacteriaceae. In addition, two approaches optimizing the identification of fastidious GNR by the MALDI Biotyper were demonstrated: formic acid-based on-target sample treatment and reductions in cutoff scores to increase the species identification rates.

Similar efficacy of broad-range ITS PCR and conventional fungal culture for diagnosing fungal infections in non-immunocompromised patients.

BACKGROUND: Broad-range fungal inter spacer region (ITS) polymerase chain reaction (PCR) has been evaluated for the detection and identification of fungi in clinical specimens from severely immunocompromised patients, but not in non-selected patients. Thus, the aim of this study was to compare the diagnostic performance of ITS PCR with that of fungal culture and to investigate its clinical impact on the diagnosis of fungal infections in non-immunocompromised patients. The corresponding patients' data were retrieved by detailed medical chart reviews. RESULTS: Results from 251 specimens showed a high concordance of 89.6 % for ITS PCR and fungal culture. The analytical sensitivity and specificity of ITS PCR considering culture as gold standard were 87.7 and 90.3 %, respectively, the positive and negative predictive value (PPV and NPV) were 76 and 95.5 %, respectively. Assessing the clinical probability of a fungal infection based on detailed chart reviews, PCR had a clinical sensitivity of 88.9 %, a specificity of 86.3 %, a PPV of 64.0 % and a NPV of 96.6 %. The overall performance of fungal broad-range PCR was similar to that of culture. CONCLUSIONS: Our data show that, in non-selected and non-immunocompromised patients, the performance of ITS PCR is similar to that of culture for detecting fungal infections, not the least because sensitivity of culture in patients under antifungal treatment is surprisingly high. Compared to culture, PCR has the advantage of a rapid time-to-result (approximately two working days), proper identification of rare pathogens, prompt initiation of a species-targeted antifungal treatment, and prospects for automation.

Determination of MIC distribution and epidemiological cutoff values for bedaquiline and delamanid in Mycobacterium tuberculosis using the MGIT 960 system equipped with TB eXiST.

Bedaquiline (Sirturo) and delamanid (Deltyba) have recently been approved by the regulatory authorities for treatment of multidrug-resistant tuberculosis (MDR-TB). Antimicrobial susceptibility testing is not established for either substance. On the basis of the use of the MGIT 960 system equipped with EpiCenter/TB eXiST, we determined a mean bedaquiline MIC for wild-type strains of 0.65 mg/liter (median, 0.4 mg/liter) and an epidemiological cutoff (ECOFF) of 1.6 mg/liter; for delamanid, a mean wild-type drug MIC of 0.013 mg/liter (median, 0.01 mg/liter) and an ECOFF of 0.04 mg/liter were determined.

Evaluation of the Rapidec Carba NP Test for Detection of Carbapenemases in Enterobacteriaceae.

This study evaluated the performance of the Rapidec Carba NP test, which was introduced recently into the market for the detection of carbapenemase production in a broad spectrum of ß-lactamase-producing Enterobacteriaceae clinical isolates. In total, 252 clinical Enterobacteriaceae isolates that had been genetically characterized with respect to carbapenemase, extended-spectrum ß-lactamase (ESBL), and AmpC genes were analyzed; 51/252 isolates (20.2%) were genetically confirmed to be carbapenemase producers, whereas 201/252 isolates (79.8%) were genetically negative for the presence of carbapenemase genes. The Rapidec Carba NP test was applied according to the manufacturer's instructions, and results were read after 30 and 120 min of incubation. The overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Rapidec Carba NP test were 90.2%, 100%, 100%, and 97.6%, respectively, when the manufacturer's instructions were followed. Four of 5 false-negative results occurred with OXA-48-like enzymes. After an incubation time of 30 min, the sensitivity was 49%. The sensitivity increased to 100% when the recommended bacterial inoculum was doubled and the test was read strictly after 120 min of incubation. The Rapidec Carba NP test is a useful tool for the reliable confirmation of carbapenemase-producing Enterobacteriaceae isolates. The test should be read strictly after 120 min of incubation and the inoculum should be larger than recommended by the manufacturer.

Evaluation of carbapenemase screening and confirmation tests with Enterobacteriaceae and development of a practical diagnostic algorithm.

Reliable identification of carbapenemase-producing members of the family Enterobacteriaceae is necessary to limit their spread. This study aimed to develop a diagnostic flow chart using phenotypic screening and confirmation tests that is suitable for implementation in different types of clinical laboratories. A total of 334 clinical Enterobacteriaceae isolates genetically characterized with respect to carbapenemase, extended-spectrum ß-lactamase (ESBL), and AmpC genes were analyzed. A total of 142/334 isolates (42.2%) were suspected of carbapenemase production, i.e., intermediate or resistant to ertapenem (ETP) and/or meropenem (MEM) and/or imipenem (IPM) according to EUCAST clinical breakpoints (CBPs). A group of 193/334 isolates (57.8%) showing susceptibility to ETP, MEM, and IPM was considered the negative-control group in this study. CLSI and EUCAST carbapenem CBPs and the new EUCAST MEM screening cutoff were evaluated as screening parameters. ETP, MEM, and IPM with or without aminophenylboronic acid (APBA) or EDTA combined-disk tests (CDTs) and the Carba NP-II test were evaluated as confirmation assays. EUCAST temocillin cutoffs were evaluated for OXA-48 detection. The EUCAST MEM screening cutoff (<25 mm) showed a sensitivity of 100%. The ETP APBA CDT on Mueller-Hinton agar containing cloxacillin (MH-CLX) displayed 100% sensitivity and specificity for class A carbapenemase confirmation. ETP and MEM EDTA CDTs showed 100% sensitivity and specificity for class B carbapenemases. Temocillin zone diameters/MIC testing on MH-CLX was highly specific for OXA-48 producers. The overall sensitivity, specificity, positive predictive value, and negative predictive value of the Carba NP-II test were 78.9, 100, 100, and 98.7%, respectively. Combining the EUCAST MEM carbapenemase screening cutoff (<25 mm), ETP (or MEM), APBA, and EDTA CDTs, and temocillin disk diffusion on MH-CLX promises excellent performance for carbapenemase detection.

Aminoglycoside-modifying enzymes determine the innate susceptibility to aminoglycoside antibiotics in rapidly growing mycobacteria.

OBJECTIVES: Infections caused by the rapidly growing mycobacterium (RGM) Mycobacterium abscessus are notoriously difficult to treat due to the innate resistance of M. abscessus to most clinically available antimicrobials. Aminoglycoside antibiotics (AGA) are a cornerstone of antimicrobial chemotherapy against M. abscessus infections, although little is known about intrinsic drug resistance mechanisms. We investigated the role of chromosomally encoded putative aminoglycoside-modifying enzymes (AME) in AGA susceptibility in M. abscessus. METHODS: Clinical isolates of M. abscessus were tested for susceptibility to a series of AGA with different substituents at positions 2', 3' and 4' of ring 1 in MIC assays. Cell-free extracts of M. abscessus type strain ATCC 19977 and Mycobacterium smegmatis strains SZ380 [aac(2')-Id(+)], EP10 [aac(2')-Id(-)] and SZ461 [aac(2')-Id(+), rrs A1408G] were investigated for AGA acetylation activity using thin-layer chromatography (TLC). Cell-free ribosome translation assays were performed to directly study drug-target interaction. RESULTS: Cell-free translation assays demonstrated that ribosomes of M. abscessus and M. smegmatis show comparable susceptibility to all tested AGA. MIC assays for M. abscessus and M. smegmatis, however, consistently showed the lowest MIC values for 2'-hydroxy-AGA as compared with 2'-amino-AGA, indicating that an aminoglycoside-2'-acetyltransferase, Aac(2'), contributes to innate AGA susceptibility. TLC experiments confirmed enzymatic activity consistent with Aac(2'). Using M. smegmatis as a model for RGM, acetyltransferase activity was shown to be up-regulated in response to AGA-induced inhibition of protein synthesis. CONCLUSIONS: Our findings point to AME as important determinants of AGA susceptibility in M. abscessus.

Lack of antimicrobial bactericidal activity in Mycobacterium abscessus.

Antibiotic therapy of infections caused by the emerging pathogen Mycobacterium abscessus is challenging due to the organism's natural resistance toward most clinically available antimicrobials. We investigated the bactericidal activity of antibiotics commonly administered in M. abscessus infections in order to better understand the poor therapeutic outcome. Time-kill curves were generated for clinical M. abscessus isolates, Mycobacterium smegmatis, and Escherichia coli by using antibiotics commonly categorized as bactericidal (amikacin and moxifloxacin) or bacteriostatic (tigecycline and linezolid). In addition, the impact of aminoglycoside-modifying enzymes on the mode of action of substrate and nonsubstrate aminoglycosides was studied by using M. smegmatis as a model organism. While amikacin and moxifloxacin were bactericidal against E. coli, none of the tested compounds showed bactericidal activity against M. abscessus. Further mechanistic investigations of the mode of action of aminoglycosides in M. smegmatis revealed that the bactericidal activity of tobramycin and gentamicin was restored by disruption of the chromosomal aac(2') gene in the mycobacterial genome. The lack of bactericidal antibiotics in currently recommended treatment regimens provides a reasonable explanation for the poor therapeutic outcome in M. abscessus infection. Our findings suggest that chromosomally encoded drug-modifying enzymes play an important role in the lack of aminoglycoside bactericidal activity against rapidly growing mycobacteria.

Evaluation of the Bruker MALDI Biotyper for identification of Gram-positive rods: development of a diagnostic algorithm for the clinical laboratory.

Reported matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification rates of Gram-positive rods (GPR) are low compared to identification rates of Gram-positive cocci. In this study, three sample preparation methods were compared for MALDI-TOF MS identification of 190 well-characterized GPR strains: direct transfer, direct transfer-formic acid preparation, and ethanol-formic acid extraction. Using the interpretation criteria recommended by the manufacturer, identification rates were significantly higher for direct transfer-formic acid preparation and ethanol-formic acid extraction than for direct transfer. Reducing the species cutoff from 2.0 to 1.7 significantly increased species identification rates. In a subsequent prospective study, 215 clinical GPR isolates were analyzed by MALDI-TOF MS, and the results were compared to those for identification using conventional methods, with discrepancies being resolved by 16S rRNA and rpoB gene analysis. Using the direct transfer-formic acid preparation and a species cutoff of 1.7, congruencies on the genus and species levels of 87.4% and 79.1%, respectively, were achieved. In addition, the rate of nonidentified isolates dropped from 12.1% to 5.6% when using an extended database, i.e., the Bruker database amended by reference spectra of the 190 GPR of the retrospective study. Our data demonstrate three ways to improve GPR identification by the Bruker MALDI Biotyper, (i) optimize sample preparation using formic acid, (ii) reduce cutoff scores for species identification, and (iii) expand the database. Based on our results, we suggest an identification algorithm for the clinical laboratory combining MALDI-TOF MS with nucleic acid sequencing.