Title Participants Abstract "Engineered endolysin-based ""artilysins"" to combat multidrug-resistant gram-negative pathogens" "Yves Briers, Victor Van Puyenbroeck, William Cenens, Abram Aertsen, Guido Volckaert, Rob Lavigne" "The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular endolysin are able to kill these (multidrug-resistant) strains in vitro with a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caenorhabditis elegans)." "Mycobactericidal effects of different regimens measured by molecular bacterial load assay among people treated for multidrug-resistant tuberculosis in Tanzania" "Peter M. Mbelele, Emmanuel A. Mpolya, Elingarami Sauli, Bariki Mtafya, Nyanda E. Ntinginya, Kennedy K. Addo, Kathy Kreppel, Sayoki Godfrey Mfinanga, Patrick P. J. Phillips, Stephen H. Gillespie, Scott K. Heysell, Wilber Sabiiti, Stellah G. Mpagama" "Rifampin or multidrug-resistant tuberculosis (RR/MDR-TB) treatment has largely transitioned to regimens free of the injectable aminoglycoside component, despite the drug class' purported bactericidal activity early in treatment. We tested whether Mycobacterium tuberculosis killing rates measured by tuberculosis molecular bacterial load assay (TB-MBLA) in sputa correlate with composition of the RR/MDRTB regimen. Serial sputa were collected from patients with RR/MDR- and drug-sensitive TB at days 0, 3, 7, and 14, and then monthly for 4 months of anti-TB treatment. TB-MBLA was used to quantify viable M. tuberculosis 16S rRNA in sputum for estimation of colony forming units per ml (eCFU/ml). M. tuberculosis killing rates were compared among regimens using nonlinear-mixed-effects modeling of repeated measures. Thirty-seven patients produced 296 serial sputa and received treatment as follows: 13 patients received an injectable bedaquiline-free reference regimen, 9 received an injectable bedaquiline-containing regimen, 8 received an all-oral bedaquiline-based regimen, and 7 patients were treated for drug-sensitive TB with conventional rifampin/isoniazid/pyrazinamide/ethambutol (RHZE). Compared to the adjusted M. tuberculosis killing of -0.17 (95% confidence interval [CI] -0.23 to -0.12) for the injectable bedaquiline-free reference regimen, the killing rates were -0.62 (95% CI -1.05 to -020) log,, eCFU/ml for the injectable bedaquiline-containing regimen (P=0.019), -0.35 (95% CI -0.65 to -0.13) log(10) eCFU/ml for the all-oral bedaquiline-based regimen (P=0.054), and -029 (95% CI -0.78 to -0.22) log(10) eCFU/ml for the RHZE regimen (P=0332). Thus, M. tuberculosis killing rates from sputa were higher among patients who received bedaquiline but were further improved with the addition of an injectable aminoglycoside." "Three promising antimycobacterial medicinal plants reviewed as potential sources of drug hit candidates against multidrug-resistant tuberculosis" "Naasson Tuyiringire, Serawit Deyno, Casim Umba Tolo, Jean-Pierre Munyampundu, Claude Mambo Muvunyi, Yvan Vander Heyden" "Regimens of current drugs for tuberculosis are lengthy and are associated with many adverse effects. Currently,the emergence of different resistant strains has been observed. This urges a need for the discovery and development of novel drugs. The main sources of drug lead candidates are based on natural products. Zanthoxylum leprieurii, Lantana camara, and Cryptolepis Sanguinolenta are among the plants that have antimycobacterial activity. Recent technological methods, such as metabolomics, can rapidly detect and identify active compounds from medicinal plants. In this review, we aim to provide an overview and discussion of the antimycobacterial activity, phytochemical analysis and toxicity profile of these plants and their products as well as the potential of metabolomic fingerprinting of medicinal plants with a given activity on microbes, in the search for the potential drug hit molecules. The information for this review was extracted from databases such as Excerpta Medica Database, Google Scholar, Springer, and PubMed Central. Primary studies, using a combination of the keywords antimycobacterial medicinal plant, multidrug-resistant tuberculosis, phytochemistry, toxicity, Zanthoxylum leprieurii, Lantana camara, Cryptolepis sanguinolenta, and plant metabolomics/metabolic fingerprinting of plant extracts, have been considered. The above-mentioned plant species showed antimycobacterial activity against drug-resistant strains of M. tuberculosis. They may provide potential candidates for novel drugs against multidrug-resistant tuberculosis. However, extensive work is still needed. To our knowledge, there is no or limited literature that reports the metabolic fingerprints of these plants. The analysis of the metabolite fingerprints of medicinal plants with similar antimicrobial activity could be important to determine whether the activity results from common metabolites within different plant species. This review shows that these plants are potential candidates to provide drug hits against multidrug-resistant tuberculosis strains. Future studies of compound optimization, in vivo safety and efficacy, as well as of the specific mechanisms of action are however required." "Management of multidrug-resistant tuberculosis with shorter treatment regimen in Niger: nationwide programmatic achievements" "Alberto Piubello, Mahamadou Bassirou Souleymane, Souleymane Hassane Harouna, Abdourahamane Yacouba, Pauline Lempens, Mourtala Mohamed Assao-Neino, Ibrahim Maman-Lawan, Sala Attaher, Boubacar Moustapha, Alphazazi Soumana, Assiatou Gagara-Issoufou, Nimer Ortuno Gutierrez, Alberto Roggi, Mourad Gumusboga, Zelika Hamidou-Harouna, Jeroen Dockx, Saidou Mamadou, Bouke de Jong, Tom Decroo, Armand Van Deun" "Background: In Niger, the Shorter Treatment Regimen (STR) has been implemented nationwide for rifampicin resistant tuberculosis (RR-TB), since 2008. No previous publication has shown the results from countrywide programmatic implementation using few exclusion criteria, nor exhaustively assessed the effect of initial resistance to companion drugs on outcomes.Methods: The National Tuberculosis Programme and the Damien Foundation conducted a retrospective observational study to evaluate the management of RR-TB from 2008 to 2016. Baseline resistance to drugs was assessed phenotypically, complemented by screening the inhA, katG and pncA genes. Cured patients were followed-up for a period of one year after cure.Findings: Among 1044 patients tested for rifampicin resistance, mainly previously treated patients, 332 were diagnosed with pulmonary RR/TB, 288 were enrolled on treatment and 255 started on STR. Six patients received a modified STR.Among 249 patients on standardised STR, 207 (83.1%) were cured relapse-free, eight (3.2%) had failure, 23 (9.2%) died, seven (2.8%) were lost to follow-up and four (1.6%) relapsed.The risk of unfavourable outcome was higher in patients with initial resistance to fluoroquinolones (aOR 20.4, 95%CI:5.6-74.6) and very severely underweight (aOR 3.9, 95%CI:1.5-10.1). Successful outcome was not affected by initial resistance to companion drugs. Serious ototoxicity was reported in eight patients (3.2%).Interpretation: A comprehensive nationwide approach to multidrug-resistant tuberculosis management using the STR was feasible and successful. Outcomes were not affected by initial resistance to companion drugs. Our study confirms the effectiveness and safety of the STR." "Late diagnosis of multidrug-resistant tuberculosis in a child at Dr George Mukhari Academic Hospital Ga-Rankuwa, South Africa; a case report" "Bakani A. Siwele, Ndivhuho A. Makhado, Matodzi T. Mariba" "Introduction: South Africa has one of the top ten tuberculosis burdens in the world, only lagging behind countries with significantly larger populations. Increased awareness of extrapulmonary tuberculosis, specifically spinal tuberculosis, is necessary, because of the HIV epidemic.Case presentation: This report describes the case of a 9-year-old male patient who was suspected of having multidrug-resistant (MDR) tuberculosis, based on failure to recover clinically and radiologically after 6 months on first-line anti-tuberculosis treatment. Pus samples were sent to an accredited academic laboratory for histopathology, microscopy, culture, line-probe assay (MTBDRplus assay) and phenotypic MGIT 960 drug susceptibility tests. Second-line MDR tuberculosis treatment was introduced. Clinical, radiological, physical processes and more laboratory tests were conducted to document whether or not there was improvement in the patient.Management and outcome: After laboratory diagnosis of MDR tuberculosis, the patient was started on MDR tuberculosis treatment. The patient started improving remarkably after the introduction of anti-tuberculosis treatment and rehabilitation, although he also required surgery to stabilise the spine. Neurological improvement was observed in the patient and he fully recovered.Discussion: Although the diagnosis of spinal MDR tuberculosis may not be achieved easily by culture, the well-known gold standard method of tuberculosis diagnosis, it is of great importance to rapidly initiate an effective anti-tuberculosis treatment of drug-resistant strains to reduce the deformity of the spine." "Detection of multidrug-resistant tuberculosis from stored DNA samples : a multicenter study" "Marie Sylvianne Rabodoarivelo, A. Brandao, M. C. Cergole Novella, A. G. C. Bombonatte, B. Imperiale, N. Rakotosamimanana, N. Morcillo, V. Rasolofo, Juan Carlos Palomino, A. Martin" "Background: In low-income countries, rapid detection of tuberculosis (TB) drug resistance is often restricted by the difficulties of transporting and storing sputum samples from remote health centers to the reference laboratories where molecular tests are available. The aim of this study was to evaluate the performance of four transport and storage systems for molecular detection of rifampicin (RIF) and isoniazid (INH) resistance. Methods: This was a multicenter study. Molecular detection of RIF and INH resistance was performed directly from smear-positive TB sputa spotted on a slide, FTA card, GenoCard, and ethanol using the Genotype MTBDRplus assay. The performance of the DNA extraction method from each storage support to detect drug resistance was assessed by calculating their sensitivity and specificity compared to the phenotypic method. Results: From all sites, the overall sensitivity and specificity for RIF-resistance detection was 88% and 85%, respectively, for slides, 86% and 92%, respectively, for GenoCard, 87% and 89%, respectively, for FTA card, and 88% and 92%, respectively, for ethanol. For INH-resistance detection, the overall sensitivity and specificity was 82% and 90%, respectively, for slides, 85% and 96%, respectively, for GenoCard, 86% and 92%, respectively, for FTA card, and 86% and 94%, respectively, for ethanol. Conclusion: Smear slides and filter cards showed to be very useful tools to facilitate DNA extraction from sputum samples with the potential to accelerate the detection of drug resistance in remote areas." "Rapid diagnosis of pyrazinamide-resistant multidrug-resistant tuberculosis using a molecular-based diagnostic algorithm" "S O Simons, T Van der Laan, A Mulder, J van Ingen, Leen Rigouts, P N R Dekhuijzen, M J Boeree, D van Soolingen" "There is an urgent need for rapid and accurate diagnosis of pyrazinamide-resistant multidrug-resistant tuberculosis (MDR-TB). No diagnostic algorithm has been validated in this population. We hypothesized that pncA sequencing added to rpoB mutation analysis can accurately identify patients with pyrazinamide-resistant MDR-TB. We identified from the Dutch national database (2007-11) patients with a positive Mycobacterium tuberculosis culture containing a mutation in the rpoB gene. In these cases, we prospectively sequenced the pncA gene. Results from the rpoB and pncA mutation analysis (pncA added to rpoB) were compared with phenotypic susceptibility testing results to rifampicin, isoniazid and pyrazinamide (reference standard) using the Mycobacterial Growth Indicator Tube 960 system. We included 83 clinical M. tuberculosis isolates containing rpoB mutations in the primary analysis. Rifampicin resistance was seen in 72 isolates (87%), isoniazid resistance in 73 isolates (88%) and MDR-TB in 65 isolates (78%). Phenotypic reference testing identified pyrazinamide-resistant MDR-TB in 31 isolates (48%). Sensitivity of pncA sequencing added to rpoB mutation analysis for detecting pyrazinamide-resistant MDR-TB was 96.8%, the specificity was 94.2%, the positive predictive value was 90.9%, the negative predictive value was 98.0%, the positive likelihood was 16.8 and the negative likelihood was 0.03. In conclusion, pyrazinamide-resistant MDR-TB can be accurately detected using pncA sequencing added to rpoB mutation analysis. We propose to include pncA sequencing in every isolate with an rpoB mutation, allowing for stratification of MDR-TB treatment according to pyrazinamide susceptibility." "Epidemiology and molecular typing of multidrug-resistant bacteria in tertiary hospitals and nursing homes in Flanders, Belgium" "Stefanie van Koeveringe, Veerle Matheeussen, Annette Schuermans, Sien De Koster, Natascha Perales Selva, Dieter De Coninck, Katrien De Bruyne, Klaas Mensaert, Marjolein Kluytmans-van den Bergh, Jan Kluytmans, Herman Goossens, Wouter Dhaeze, Isabel Leroux-Roels" "This study aimed to map MDRO carriage and potential transmission within and between three Flemish tertiary care hospitals and their neighbouring nursing homes. A cross-sectional MDRO prevalence survey was organized between October 2017 and February 2019. Perianal swabs were cultured for detection of MDRO. Determination of clonal relatedness based on wgMLST allelic profiles was performed. The prevalence of MDRO in Belgian hospitals and NHs is on the rise, compared to previous studies, and transmission in and between institutions is observed. These results re-emphasize the need for a healthcare network-wide infection prevention strategy in which WGS of MDRO strains can be supportive." "Assessment of colistin heteroresistance among multidrug-resistant Klebsiella pneumoniae isolated from intensive care patients in Europe" "Sahaya Glingston Rajakani, Adwoa Sey, Mariem El Bounja, Gérald Glupczynski, Surbhi Malhotra" "Heteroresistance (HR) to colistin is especially concerning in settings where multi-drug-resistant (MDR) K. pneumoniae are prevalent and empiric use of colistin might lead to treatment failures. This study aimed to assess the frequency of occurrence of colistin HR (CHR) among (MDR) K. pneumoniae (n = 676) isolated from patients hospitalized in 13 intensive care units (ICUs) in six European countries in a clinical trial assessing the impact of decolonization strategies. All isolates were whole-genome-sequenced and studied for in vitro colistin susceptibility. The majority were colistin-susceptible (CS) (n = 597, MIC ≤ 2 µg/mL), and 79 were fully colistin-resistant (CR) (MIC > 2 µg/mL). A total of 288 CS isolates were randomly selected for population analysis profiling (PAP) to assess CHR prevalence. CHR was detected in 108/288 CS K. pneumoniae. No significant association was found between the occurrence of CHR and country, MIC-value, K-antigen type, and O-antigen type. Overall, 92% (617/671) of the K. pneumoniae were MDR with high prevalence among CS (91%, 539/592) and CR (98.7%, 78/79) isolates. In contrast, the proportion of carbapenemase-producing K. pneumoniae (CP-Kpn) was higher among CR (72.2%, 57/79) than CS isolates (29.3%, 174/594). The proportions of MDR and CP-Kpn were similar among CHR (MDR: 85%, 91/107; CP-Kpn: 29.9%, 32/107) and selected CS isolates (MDR: 84.7%, 244/288; CP-Kpn: 28.1%, 80/285). WGS analysis of PAP isolates showed diverse insertion elements in mgrB or even among technical replicates underscoring the stochasticity of the CHR phenotype. CHR isolates showed high sequence type (ST) diversity (Simpson’s diversity index, SDI: 0.97, in 52 of the 85 STs tested). CR (SDI: 0.85) isolates were highly associated with specific STs (ST101, ST147, ST258/ST512, p ≤ 0.003). The widespread nature of CHR among MDR K. pneumoniae in our study urge the development of rapid HR detection methods to inform on the need for combination regimens." "Multicenter interlaboratory study of routine systems for the susceptibility testing of temocillin using a challenge panel of multidrug-resistant strains" "Stefanie Desmet" "Accurate susceptibility result of temocillin (TMO) is important for treating infections caused by multidrug-resistant Enterobacterales. This multicenter study aimed to investigate the performance of routine temocillin testing assays against Enterobacterales challenging strains. Forty-seven selected clinical isolates were blindly analyzed by 12 Belgian laboratories using VITEK® 2 (n = 5) and BD Phoenix™ (n = 3) automated systems, ETEST® gradient strip (n = 3), and disk (3 brands) diffusion method (DD; n = 6) for temocillin susceptibility using standardized methodology. Results were interpreted using EUCAST 2023 criteria and compared to the broth microdilution (BMD; Sensititre™ panel) method used as gold standard. Methods' reproducibility was assessed by testing 3 reference strains in triplicate. A total of 702 organism-drug results were obtained against 33 TMO-susceptible and 14 TMO-resistant isolates. Excluding Proteae species (P. mirabilis and M. morganii), the essential agreement rates were excellent (91.5-100%) for all MIC-based methods. The highest category agreement was achieved by ETEST® (97.5%) followed by VITEK® 2 (93.2%), disk diffusion (91.6%), and BD Phoenix™ (88.5%). BD Phoenix™ and paper disk diffusion overcalled resistance (11.5% and 6.8% of major discrepancies, respectively), while ROSCO tablets diffusion and VITEK® 2 generated higher very major discrepancies (7.1% and 4.2% respectively). Inter-assay reproducibility was unsatisfactory using recommended E. coli ATCC 25922 strain but was excellent with E. coli ATCC 35218 and K. pneumoniae ATCC 700603 strains. This interlaboratory study suggests that routine testing methods provide accurate and reproducible TMO categorization results except for Proteae species."