Panton-Valentine Leukocidin-Positive Staphylococcus aureus in Family and Pet Cat.

Continued detection of Panton-Valentine leukocidin-positive Staphylococcus aureus in samples from a family with severe repeated skin infections and their pet cat suggests transmission between the family and the cat. Decolonizing the pet led to successful elimination of the bacteria from the household. Clinicians should consider pet cats as possible reinfection sources.

Tandem amplification of a plasmid-borne tet(A) variant gene confers tigecycline resistance in Escherichia coli.

OBJECTIVES: To elucidate the mechanism of tigecycline resistance in Escherichia coli that is mediated by the tet(A) variant gene. METHODS: E. coli strain 573 carried a plasmid-borne tet(A) variant gene, tentatively designated tet(A)TIG, that conferred decreased tigecycline susceptibility (MIC 0.5 mg/L). When exposed to increasing concentrations of tigecycline (0.25-8 mg/L), mutants growing at 2, 4 and 8 mg/L were obtained and sequenced. Copies of plasmid and tet(A)TIG relative to the chromosomal DNA in the mutants were determined by WGS and quantitative PCR (qPCR). Expression of tet(A)TIG in the mutants was evaluated by RT-qPCR. The tet(A)TIG-carrying plasmids were visualized by S1-PFGE and Southern blot hybridization. PCR served for the detection of a tet(A)TIG-carrying unconventional circularizable structure (UCS). RESULTS: Tigecycline resistance with maximum MICs of 16 mg/L was seen in E. coli mutants selected in the presence of tigecycline. Compared with the parental strain, the relative copy number and transcription level of tet(A)TIG in the mutants increased significantly in the presence of 2, 4 and 8 mg/L tigecycline, respectively. With increasing tigecycline selection pressure, the tet(A)TIG-carrying plasmids in the mutants increased in size, correlating with the number of tandem amplificates of a ΔTnAs1-flanked UCS harbouring tet(A)TIG. These tandem amplificates were not stable in the absence of tigecycline. CONCLUSIONS: Tigecycline resistance is due to the tandem amplification of a ΔTnAs1-flanked tet(A)TIG-carrying plasmid-borne segment in E. coli. The gain/loss of the tandem amplificates in the presence/absence of tigecycline represents an economic way for the bacteria to survive in the presence of tigecycline.

Global spread of the linezolid-resistant Enterococcus faecalis ST476 clonal lineage carrying optrA.

OBJECTIVES: To investigate the global distribution of an optrA-harbouring linezolid-resistant Enterococcus faecalis ST476 clonal lineage. METHODS: Comprehensive searches of the NCBI database were performed to identify published peer-reviewed articles and genomes of E. faecalis ST476. Each genome was analysed for resistome, virulome, OptrA variant and optrA genetic contexts. A phylogenetic comparison of ST476 genomes with publicly available genomes of other STs was also performed. RESULTS: Sixty-six E. faecalis ST476 isolates from 15 countries (China, Japan, South Korea, Austria, Denmark, Spain, Czech Republic, Colombia, Tunisia, Italy, Malaysia, Belgium, Germany, United Arab Emirates and Switzerland) mainly of human and animal origin were identified. Thirty available ST476 genomes compared with genomes of 591 STs indicated a progressive radiation of E. faecalis STs starting from ST21. The closest ancestral node for ST476 was ST1238. Thirty E. faecalis ST476 genomes exhibited 3-916 SNP differences. Several antimicrobial resistance and virulence genes were conserved among the ST476 genomes. The optrA genetic context exhibited a high degree of or complete identity to the chromosomal transposon Tn6674. Only three isolates displayed an optrA-carrying plasmid with complete or partial Tn6674. The WT OptrA protein was most widespread in the ST476 lineage. CONCLUSIONS: Linezolid-resistant optrA-carrying E. faecalis of the clonal lineage ST476 is globally distributed in human, animal and environmental settings. The presence of such an emerging clone can be of great concern for public health. Thus, a One Health approach is needed to counteract the spread and the evolution of this enterococcal clonal lineage.

Bidirectional transfer of human cytomegalovirus strains in donor and recipient seropositive lung transplant patients.

Donor and recipient human cytomegalovirus (HCMV) seropositive (D+R+) lung transplant recipients (LTRs) often harbor multiple strains of HCMV, likely due to transmitted donor (D) strains and reactivated recipient (R) strains. To date, the extent and timely occurrence of each likely source in shaping the post-transplantation (post-Tx) strain population is unknown. Here, we deciphered the D and R origin of the post-Tx HCMV strain composition in blood, bronchoalveolar lavage (BAL), and CD45+ BAL cell subsets. We investigated either D and/or R formalin-fixed paraffin-embedded blocks or fresh D lung tissue from four D+R+ LTRs obtained before transplantation. HCMV strains were characterized by short amplicon deep sequencing. In two LTRs, we show that the transplanted lung is reseeded by R strains within the first 6 months after transplantation, likely by infiltrating CD14+ CD163+/- alveolar macrophages. In three LTRs, we demonstrate both rapid D-strain dissemination and persistence in the transplanted lung for >1 year post-Tx. Broad inter-host diversity contrasts with intra-host genotype sequence stability upon transmission, during follow-up and across compartments. In D+R+ LTRs, HCMV strains of both, D and R origin can emerge first and dominate long-term in subsequent episodes of infection, indicating replication of both sources despite pre-existing immunity.

Diversity of Staphylococcus aureus isolated from nares of ruminants.

AIMS: To examine the diversity of Staphylococcus aureus isolated from nasal swabs of ruminants in Rwanda. METHODS AND RESULTS: A total of 454 nasal swabs from 203 cows, 170 goats, and 81 sheep were examined for the presence of S. aureus, and 30 S. aureus isolates were detected and characterized pheno- and genotypically. Resistance to penicillin and/or tetracycline was observed. The isolates were assigned to eight different spa types (t21057 (novel), t10103, t18853, t20842, t318, t355, t458, and t9432) belonging to six clonal complexes (CCs) (CC152, CC30, CC3591, CC3666, CC522, and CC97). Panton-Valentine leukocidin (PVL) genes (lukF-PV/lukS-PV), the bovine leukocidin genes (lukM/lukF-P83), and the human and bovine variants of the toxic shock syndrome toxin gene tst-1 variants were detected. CONCLUSION: These findings demonstrate that the nares of ruminants in Rwanda are colonized with mastitis-associated S. aureus, including lineages that are also carried by humans, underscoring the zoonotic risk, especially for livestock keepers. These results highlight the crucial importance of hygiene measures when handling livestock.

Computed Tomography-Based Body Composition is Related to Perioperative Morbidity in Older Lung Transplant Recipients.

BACKGROUND: In older patients, a limited physical reserve is considered a contraindication for lung transplantation (LTx). Herein, we aimed to establish a computed tomography (CT)-based quantification of physical reserve in older patients scheduled for transplantation. METHODS: This retrospective study included patients older than 60 years who received LTx. Semiautomatic measurements of the mediastinal fat area and the dorsal muscle group area in pretransplantation CT scans were performed, and normalized data were correlated with clinical parameters. RESULTS: Patients (n = 108) were assigned into three groups (Musclehighfatlow [n = 25], Musclelowfathigh [n = 24], and other combinations [n = 59]). The Musclelowfathigh group had a significantly increased risk of wound infections (p = 0.002) and tracheostomy (p = 0.001) compared with Musclehighfatlow patients. The median length of intensive care unit stay (25 vs. 3.5 days; p = 0.002) and the median length of hospital stay (44 vs. 22.5 days; p = 0.013) post-LTx were significantly prolonged in the Musclelowfathigh group. Significantly more patients in this group had a prolonged ventilation time (11 vs. 0; p < 0.001). CONCLUSION: Body composition parameters determined in pretransplant chest CT scans in older LTx candidates might aid in identifying high-risk patients with a worse perioperative outcome after LTx.

Identification of ISVlu1-derived translocatable units containing optrA and/or fexA genes generated by homologous or illegitimate recombination in Lactococcus garvieae of porcine origin.

The optrA gene encodes an ABC-F protein which confers cross-resistance to oxazolidinones and phenicols. Insertion sequence ISVlu1, a novel ISL3-family member, was recently reported to be involved in the transmission of optrA in Vagococcus lutrae. However, the role of ISVlu1 in mobilizing resistance genes has not yet fully explored. In this study, two complete and three truncated copies of ISVlu1 were found on plasmid pBN62-optrA from Lactococcus garvieae. Analysis of the genetic context showed that both optrA and the phenicols resistance gene fexA were flanked by the complete or truncated ISVlu1 copies. Moreover, three different-sized ISVlu1-based translocatable units (TUs) carrying optrA and/or fexA, were detected from pBN62-optrA. Sequence analysis revealed that the TU-optrA was generated by homologous recombination while TU-fexA and TU-optrA+fexA were the products of illegitimate recombinations. Importantly, conjugation assays confirmed that pBN62-optrA was able to successfully transfer into the recipient Enterococcus faecalis JH2-2. To our knowledge, this is the first report about an optrA-carrying plasmid in L. garvieae which could horizontally transfer into other species. More importantly, the ISVlu1-flanked genetic structures containing optrA and/or fexA were also observed in bacteria of different species, which underlines that ISVlu1 is highly active and plays a vital role in the transfer of some important resistance genes, such as optrA and fexA.

Macrolide resistance in Mannheimia haemolytica isolates associated with bovine respiratory disease from the German national resistance monitoring program GERM-Vet 2009 to 2020.

Data collected from the German national resistance monitoring program GERM-Vet showed slowly increasing prevalence of macrolide resistance among bovine respiratory disease (BRD)-associated Pasteurellacae from cattle over the last decade. The focus of this study was to analyze the genetic basis of antimicrobial resistance (AMR) and the prevalence of multidrug-resistance (MDR)-mediating integrative and conjugative elements (ICEs) in 13 German BRD-associated Mannheimia haemolytica isolates collected between 2009 and 2020 via whole-genome sequencing. Antimicrobial susceptibility testing (AST) was performed via broth microdilution according to the recommendations of the Clinical and Laboratory Standards Institute for the macrolides erythromycin, tilmicosin, tulathromycin, gamithromycin, tildipirosin, and tylosin as well as 25 other antimicrobial agents. All isolates either had elevated MICs or were resistant to at least one of the macrolides tested. Analysis of whole-genome sequences obtained by hybrid assembly of Illumina MiSeq and Oxford Nanopore MinION reads revealed the presence of seven novel Tn7406-like ICEs, designated Tn7694, and Tn7724- Tn7729. These ICEs harbored the antimicrobial resistance genes erm(T), mef (C), mph(G), floR, catA3, aad(3")(9), aph(3')-Ia, aac(3)-IIa, strA, strB, tet(Y), and sul2 in different combinations. In addition, mutational changes conferring resistance to macrolides, nalidixic acid or streptomycin, respectively, were detected among the M. haemolytica isolates. In addition, four isolates carried a 4,613-bp plasmid with the ß-lactamase gene blaROB - 1. The detection of the macrolide resistance genes erm(T), mef (C), and mph(G) together with other resistance genes on MDR-mediating ICEs in bovine M. haemolytica may explain the occurrence of therapeutic failure when treating BRD with regularly used antimicrobial agents, such as phenicols, penicillins, tetracyclines, or macrolides. Finally, pathogen identification and subsequent AST is essential to ensure the efficacy of the antimicrobial agents applied to control BRD in cattle.

Risk Communication on Zoonoses and Antimicrobial Resistance-How Do Exotic Pet Owners Perceive the Communication of Their Veterinarians?

Exotic animals traded and kept as pets can transmit a variety of diseases to humans and other animals, and vice versa. Therefore, it is essential for pet owners, particularly vulnerable groups, to be informed about associated risks. Veterinarians play a crucial role in informing pet owners about health risks associated with zoonotic pathogens and antimicrobial resistance (AMR) and should, therefore, have good communication skills to effectively transfer information to pet owners. Thus, exotic pet owners in Germany were surveyed on animal husbandry, veterinary consultation and risk communication. To evaluate the perception of communication, a self-developed questionnaire was used to derive a communication score. The perception of veterinarian communication received a high average score showing a high level of satisfaction. The duration of the veterinarian-client relationship was associated with better communication perception, and the frequency of communication on zoonoses and AMR was associated with the presence of a permanent veterinarian. However, the results indicated that the frequency of disseminated information on zoonoses and/or AMR from veterinarians was lower than desired by the pet owners. Therefore, more educational material on zoonoses and AMR should be made available, and the awareness concerning risk communication should be increased by further education and training at universities.

Fate of florfenicol and linezolid resistance genes and their bacterial hosts during two waste treatment models in swine feedlots.

Florfenicol resistance genes (FRGs) are widely present in livestock farms. The aim of this study was to evaluate the removal efficiencies of FRGs as well as the relationships between FRGs, mobile genetic elements (MGEs) and bacterial communities during the natural drying (ND) and anaerobic digestion (AD) processes of manure treatment in swine farms by combining bacterial isolation, quantitative PCR and metagenomic approaches. Solid manure showed a higher abundance of FRGs than fresh manure and was the main contamination source of fexA and fexB in ND farms, whilst biogas slurry displayed a lower abundance of FRGs than the wastewater in AD farms. Moreover, fresh manure and wastewater showed a high abundance of optrA, and wastewater was the main contamination source of cfr in both ND and AD farms. Both optrA/fexA-positive enterococci and cfr/fexA-positive staphylococci were mainly isolated along the farms' treatment processes. The cfr-positive staphylococci were highly prevalent in wastewater (57.14 % - 100 %) and may be associated with nasal-derived cfr-positive porcine staphylococci. An increased abundance of Enterococcus, Jeotgalibaca and Vagococcus in the bacterial community structures may account for the high optrA abundance in wastewater and Jeotgalibaca may be another potential host of optrA. Furthermore, the abundance of FRG-related MGEs increased by 22.63 % after the ND process and decreased by 66.96 % in AD farms. A significant correlation was observed between cfr and ISEnfa4, whereas no significance was found between optrA and IS1216E, although IS1216E is the predominant insertion sequence involved in the transfer of optrA. In conclusion, manure and wastewater represented independent pollution sources of FRGs in swine farms. Associated MGEs might play a key role in the transfer and persistence of FRGs. The AD process was more efficient in the removal of FRGs than the ND method, nevertheless a longer storage of slurry may be required for a complete removal.

In Vitro Antibacterial Activity of Microbial Natural Products against Bacterial Pathogens of Veterinary and Zoonotic Relevance.

Antimicrobial resistance (AMR) is considered one of the greatest threats to both human and animal health. Efforts to address AMR include implementing antimicrobial stewardship programs and introducing alternative treatment options. Nevertheless, effective treatment of infectious diseases caused by bacteria will still require the identification and development of new antimicrobial agents. Eight different natural products were tested for antimicrobial activity against seven pathogenic bacterial species (Brachyspira sp., Chlamydia sp., Clostridioides sp., Mannheimia sp., Mycobacterium sp., Mycoplasma sp., Pasteurella sp.). In a first pre-screening, most compounds (five out of eight) inhibited bacterial growth only at high concentrations, but three natural products (celastramycin A [CA], closthioamide [CT], maduranic acid [MA]) displayed activity at concentrations <2 µg/mL against Pasteurella sp. and two of them (CA and CT) also against Mannheimia sp. Those results were confirmed by testing a larger collection of isolates encompassing 64 Pasteurella and 56 Mannheimia field isolates originating from pigs or cattle, which yielded MIC90 values of 0.5, 0.5, and 2 µg/mL against Pasteurella and 0.5, 4, and >16 µg/mL against Mannheimia for CA, CT, and MA, respectively. CA, CT, and MA exhibited higher MIC50 and MIC90 values against Pasteurella isolates with a known AMR phenotype against commonly used therapeutic antimicrobial agents than against isolates with unknown AMR profiles. This study demonstrates the importance of whole-cell antibacterial screening of natural products to identify promising scaffolds with broad- or narrow-spectrum antimicrobial activity against important Gram-negative veterinary pathogens with zoonotic potential.

Global transmission of extended-spectrum cephalosporin resistance in Escherichia coli driven by epidemic plasmids.

BACKGROUND: Extended-spectrum cephalosporins (ESCs) are third and fourth generation cephalosporin antimicrobials used in humans and animals to treat infections due to multidrug-resistant (MDR) bacteria. Resistance to ESCs (ESC-R) in Enterobacterales is predominantly due to the production of extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases (AmpCs). The dynamics of ESBLs and AmpCs are changing across countries and host species, the result of global transmission of ESC-R genes. Plasmids are known to play a key role in this dissemination, but the relative importance of different types of plasmids is not fully understood. METHODS: In this study, Escherichia coli with the major ESC-R genes blaCTX-M-1, blaCTX-M-15, blaCTX-M-14 (ESBLs) and blaCMY-2 (AmpC), were selected from diverse host species and other sources across Canada, France and Germany, collected between 2003 and 2017. To examine in detail the vehicles of transmission of the ESC-R genes, long- and short-read sequences were generated to obtain complete contiguous chromosome and plasmid sequences (n = 192 ESC-R E. coli). The types, gene composition and genetic relatedness of these plasmids were investigated, along with association with isolate year, source and geographical origin, and put in context with publicly available plasmid sequences. FINDINGS: We identified five epidemic resistance plasmid subtypes with distinct genetic properties that are associated with the global dissemination of ESC-R genes across multiple E. coli lineages and host species. The IncI1 pST3 blaCTX-M-1 plasmid subtype was found in more diverse sources than the other main plasmid subtypes, whereas IncI1 pST12 blaCMY-2 was more frequent in Canadian and German human and chicken isolates. Clonal expansion also contributed to the dissemination of the IncI1 pST12 blaCMY-2 plasmid in ST131 and ST117 E. coli harbouring this plasmid. The IncI1 pST2 blaCMY-2 subtype was predominant in isolates from humans in France, while the IncF F31:A4:B1 blaCTX-M-15 and F2:A-:B- blaCTX-M-14 plasmid subtypes were frequent in human and cattle isolates across multiple countries. Beyond their epidemic nature with respect to ESC-R genes, in our collection almost all IncI1 pST3 blaCTX-M-1 and IncF F31:A4:B1 blaCTX-M-15 epidemic plasmids also carried multiple antimicrobial resistance (AMR) genes conferring resistance to other antimicrobial classes. Finally, we found genetic signatures in the regions surrounding specific ESC-R genes, identifying the predominant mechanisms of ESC-R gene movement, and using publicly available databases, we identified these epidemic plasmids from widespread bacterial species, host species, countries and continents. INTERPRETATION: We provide evidence that epidemic resistance plasmid subtypes contribute to the global dissemination of ESC-R genes, and in addition, some of these epidemic plasmids confer resistance to multiple other antimicrobial classes. The success of these plasmids suggests that they may have a fitness advantage over other plasmid types and subtypes. Identification and understanding of the vehicles of AMR transmission are crucial to develop and target strategies and interventions to reduce the spread of AMR. FUNDING: This project was supported by the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR), through the Medical Research Council (MRC, MR/R000948/1), the Canadian Institutes of Health Research (CFC-150770), and the Genomics Research and Development Initiative (Government of Canada), the German Federal Ministry of Education and Research (BMBF) grant no. 01KI1709, the French Agency for food environmental and occupational health & safety (Anses), and the French National Reference Center (CNR) for antimicrobial resistance. Support was also provided by the Biotechnology and Biological Sciences Research Council (BBSRC) through the BBSRC Institute Strategic Programme Microbes in the Food ChainBB/R012504/1 and its constituent project BBS/E/F/000PR10348 (Theme 1, Epidemiology and Evolution of Pathogens in the Food Chain).

Molecular Characterization of Chimeric Staphylococcus aureus Strains from Waterfowl.

Staphylococcus aureus is a versatile pathogen that does not only occur in humans but also in various wild and domestic animals, including several avian species. When characterizing S. aureus isolates from waterfowl, isolates were identified as atypical CC133 by DNA microarray analysis. They differed from previously sequenced CC133 strains in the presence of the collagen adhesin gene cna; some also showed a different capsule type and a deviant spa type. Thus, they were subjected to whole-genome sequencing. This revealed multiple insertions of large regions of DNA from other S. aureus lineages into a CC133-derived backbone genome. Three distinct strains were identified based on the size and extent of these inserts. One strain comprised two small inserts of foreign DNA up- and downstream of oriC; one of about 7000 nt or 0.25% originated from CC692 and the other, at ca. 38,000 nt or 1.3% slightly larger one was of CC522 provenance. The second strain carried a larger CC692 insert (nearly 257,000 nt or 10% of the strain's genome), and its CC522-derived insert was also larger, at about 53,500 nt or 2% of the genome). The third strain carried an identical CC692-derived region (in which the same mutations were observed as in the second strain), but it had a considerably larger CC522-like insertion of about 167,000 nt or 5.9% of the genome. Both isolates of the first, and two out of four isolates of the second strain also harbored a hemolysin-beta-integrating prophage carrying "bird-specific" virulence factors, ornithine cyclodeaminase D0K6J8 and a putative protease D0K6J9. Furthermore, isolates had two different variants of SCC elements that lacked mecA/mecC genes. These findings highlight the role of horizontal gene transfer in the evolution of S. aureus facilitated by SCC elements, by phages, and by a yet undescribed mechanism for large-scale exchange of core genomic DNA.

Genomic study of European Clostridioides difficile ribotype 002/sequence type 8.

Clostridioides difficile has significant clinical importance as a leading cause of healthcare-associated infections, with symptoms ranging from mild diarrhoea to severe colitis, and possible life-threatening complications. C. difficile ribotype (RT) 002, mainly associated with MLST sequence type (ST) 8, is one of the most common RTs found in humans. This study aimed at investigating the genetic characteristics of 537 C. difficile genomes of ST8/RT002. To this end, we sequenced 298 C. difficile strains representing a new European genome collection, with strains from Germany, Denmark, France and Portugal. These sequences were analysed against a global dataset consisting of 1,437 ST8 genomes available through Enterobase. Our results showed close genetic relatedness among the studied ST8 genomes, a diverse array of antimicrobial resistance (AMR) genes and the presence of multiple mobile elements. Notably, the pangenome analysis revealed an open genomic structure. ST8 shows relatively low overall variation. Thus, clonal isolates were found across different One Health sectors (humans, animals, environment and food), time periods, and geographical locations, suggesting the lineage's stability and a universal environmental source. Importantly, this stability did not hinder the acquisition of AMR genes, emphasizing the adaptability of this bacterium to different selective pressures. Although only 2.4 % (41/1,735) of the studied genomes originated from non-human sources, such as animals, food, or the environment, we identified 9 cross-sectoral core genome multilocus sequence typing (cgMLST) clusters. Our study highlights the importance of ST8 as a prominent lineage of C. difficile with critical implications in the context of One Health. In addition, these findings strongly support the need for continued surveillance and investigation of non-human samples to gain a more comprehensive understanding of the epidemiology of C. difficile.

Diversity of Staphylococcus aureus associated with mastitis from dairy cows in Rwanda.

OBJECTIVES: The objective of the present study was to examine the diversity of Staphylococcus aureus from mastitis milk samples of cows in Rwanda. METHODS: A total of 1080 quarter milk samples from 279 dairy cows were collected in 80 different farms from all five provinces of Rwanda. In total, 135 S. aureus isolates were obtained and subjected to genotyping (spa typing, DNA microarray, whole-genome sequencing (WGS)), antimicrobial susceptibility testing (AST) and phenotypic profiling by Fourier Transform Infrared (FTIR) spectroscopy (including capsular serotyping). RESULTS: Resistance to penicillin and/or tetracycline was most frequently observed. Ten sequence types (STs) (ST1, ST151, ST152, ST5477, ST700, ST7110, ST7983, ST7984, ST8320, ST97) belonging to seven clonal complexes (CCs) (CC1, CC130, CC152, CC3591, CC3666, CC705, CC97) were detected. The Panton-Valentine leukocidin (PVL) genes (lukF-PV/lukS-PV), the bovine leukocidin genes (lukM/lukF-P83) and the human and bovine toxic shock syndrome toxin gene tst-1 variants were detected. FTIR-based capsular serotyping showed CC-specific differences. Most CC97 (cap5 allele) isolates were primarily nonencapsulated (82%), whereas isolates of CC3591 and CC3666 (cap8 allele) were mostly encapsulated (86.4% and 57.8%, respectively). Our results underline the widespread global distribution of cattle-adapted CC97. CONCLUSION: The presence of CC3591 and CC3666 in bovine mastitis suggests an important role in cattle health and dairy production in Rwanda. The results of the present study support the need for a rigorous One-Health Surveillance program of the bovine-human interface.

Evaluation of a Method for Standardized Antimicrobial Susceptibility Testing with Mycoplasma hyorhinis Field Isolates.

Organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee of Antimicrobial Susceptibility Testing (EUCAST) provide standardized methodologies for antimicrobial susceptibility testing of a wide range of nonfastidious and fastidious bacteria, but so far not for Mycoplasma spp. of animal origin. Recently, a proposed method for the standardized broth microdilution testing of Mycoplasma hyorhinis using commercial Sensititre microtiter plates was presented. In this study, we evaluated this broth microdilution method with 37 field isolates and tested their susceptibility toward the following antimicrobial agents: doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin. The isolates originated from different countries, isolation sites, and years. The broth microdilution method was carried out using a modified Friis broth as the culture and test medium. For macrolides and lincosamides, a bimodal distribution with elevated MIC values could be observed for almost half of the tested field isolates, deducing reduced susceptibility toward these substances. With a recently published protocol, we were able to test a variety of field isolates, and consistent data could be obtained. Using this method, monitoring studies of Mycoplasma hyorhinis isolates can be carried out in a comparable manner, and the observed susceptibility profiles can be screened for possible changes in MIC values in the future.

Extension of Cold Static Donor Lung Preservation at 10°C.

BACKGROUND: Lung transplantation is performed on a 24/7 schedule to minimize organ ischemic time. Recent preclinical studies demonstrated superior graft preservation at 10°C compared with storage in an ice cooler (gold standard). METHODS: In this prospective, multicenter, nonrandomized clinical trial, we studied transplants from donors with overnight cross-clamp times (6:00 p.m. to 4:00 a.m.) that had an earliest allowed starting time of 6:00 a.m. Lungs meeting criteria for transplantation were retrieved, transported, and immediately transferred to a 10°C temperature-controlled incubator until implantation; 70 patients and 140 matched controls were included in this study. RESULTS: Total preservation times for lungs in the study group were 12 hours, 28 minutes (interquartile range, 10 hours, 14 minutes to 14 hours, 12 minutes) and 14 hours, 9 minutes (interquartile range, 12 hours, 3 minutes to 15 hours, 45 minutes) for the first and second lung implanted, respectively. Primary graft dysfunction grade 3 at 72 hours (primary outcome) was 5.7% in the study group versus 9.3% in matched controls (difference, −3.6; 95% confidence interval [CI], −10.5 to 5.3). No meaningful differences were observed in the need for postoperative extracorporeal membrane oxygenation (5.7 vs. 9.3%), median intensive care unit stay (5 vs. 5 days), or median hospital stay (25 vs. 30 days) between the two groups. One-year Kaplan­Meier survival was similar between the two groups (94 vs. 87%; hazard ratio, 0.65; 95% CI, 0.26 to 1.6). CONCLUSIONS: Extension of cold static preservation times at 10°C appears to be safe and has the potential to improve transplantation logistics and performance. (Funded by the UHN Foundation; Clinicaltrials.gov number, NCT04616365).

A fast and simple assay to quantify bacterial leukotoxin activity

Background: Mannheimia haemolytica is the primary bacterial pathogen in causing bovine respiratory disease with tremendous annual losses in the cattle industry. The leukotoxin from M. haemolytica is the predominant virulence factor. Several leukotoxin activity assays are available but not standardized regarding sample preparation and cell line. Furthermore, these assays suffer from a high standard error, a prolonged time consumption and often complex sample pretreatments, which is important from the bioprocess engineering point of view. Results: Within this study, an activity assay based on the continuous cell line BL3.1 combined with a commercial available adenosine triphosphate viability assay kit was established. The leukotoxin activity was found to be strongly dependent on the sample preparation. Furthermore, the interfering effect of lipopolysaccharides in the sample could be successfully suppressed by adding polymyxin B. We reached a maximum relative P95 value of 14%, which is more than seven times lower compared to current available assays as well as a time reduction up to 88%. Conclusion: Ultimately, the established leukotoxin activity assay is simple, fast and has a high reproducibility. Critical parameters regarding the sample preparation were characterized and optimized making complex sample purification superfluous.