Recent Isolates Research Articles

Molecular detection and phylogenetic analysis of newly emerging foot-and-mouth disease virus type A, Lineage EURO-SA in Egypt in 2022

A newly emerging and exotic foot-and-mouth disease virus (FMDV) caused a recent outbreak of serotype A in Egypt in 2022, which affected cattle and water buffalo. Previous phylogenetic studies on FMDV circulating in Egypt have mainly focused on genomic regions encoding the structural proteins which determine FMDV serotype. No study has yet determined structural proteins sequences of the newly emerging Europe-South America (EURO-SA) lineage which was recently isolated from Egypt during a routine surveillance in 2022. The objective of the current study was to analyze the structural proteins of the Venezuelan type which belongs to EURO-SA. The new isolate was related to serotype A lineage Euro-South America. Phylogentic analyses have reveled that the newly isolated lineage samples were closely related to reported sequences that have been identified in Venzuela and Colombia. Analysis of structural protein sequences revealed the recent isolates belong to prototype strain A24 Cruzeiro. Notably, nucleotide sequences of the Egyptian isolate was related to Venezuelan, Brazilian, and Colombian strains with identity not exceeding 90%. The divergence which appears in the genetic identity of the Egyptian A/EURO-SA lineage from other related strains may be attributed to the absence of Euro-SA lineage sequence from Egypt. The present study is the first report on the detection of EURO-SA lineage in Egypt. The recent detection of the EURO-SA lineage samples may be explained due to imported animals from Colombia or Brazil which share geographical borders with Venezuela. The findings of the present study highlight the significance of continuous monitoring of FMDV in Egypt for newly emerging FMDVs.

Divergent Evolutionary Pathways of Myxoma Virus in Australia: Virulence Phenotypes in Susceptible and Partially Resistant Rabbits Indicate Possible Selection for Transmissibility.

ABSTRACTTo characterize the ongoing evolution of myxoma virus in Australian rabbits, we used experimental infections of laboratory rabbits to determine the virulence and disease phenotypes of recent virus isolates. The viruses, collected between 2012 and 2015, fell into three lineages, one of which, lineage c, experienced a punctuated increase in evolutionary rate. All viruses were capable of causing acute death with aspects of neutropenic septicemia, characterized by minimal signs of myxomatosis, the occurrence of pulmonary edema and bacteria invasions throughout internal organs, but with no inflammatory response. For the viruses of highest virulence all rabbits usually died at this point. In more attenuated viruses, some rabbits died acutely, while others developed an amyxomatous phenotype. Rabbits that survived for longer periods developed greatly swollen cutaneous tissues with very high virus titers. This was particularly true of lineage c viruses. Unexpectedly, we identified a line of laboratory rabbits with some innate resistance to myxomatosis and used these in direct comparisons with the fully susceptible rabbit line. Importantly, the same disease phenotype occurred in both susceptible and resistant rabbits, although virulence was shifted toward more attenuated grades in resistant animals. We propose that selection against inflammation at cutaneous sites prolongs virus replication and enhances transmission, leading to the amyxomatous phenotype. In some virus backgrounds this creates an immunosuppressive state that predisposes to high virulence and acute death. The alterations in disease pathogenesis, particularly the overwhelming bacterial invasions that characterize the modern viruses, suggest that their virulence grades are not directly comparable with earlier studies.IMPORTANCE The evolution of the myxoma virus (MYXV) following its release as a biological control for European rabbits in Australia is the textbook example of the coevolution of virus virulence and host resistance. However, most of our knowledge of MYXV evolution only covers the first few decades of its spread in Australia and often with little direct connection between how changes in virus phenotype relate to those in the underlying virus genotype. By conducting detailed experimental infections of recent isolates of MYXV in different lines of laboratory rabbits, we examined the ongoing evolution of MYXV disease phenotypes. Our results reveal a wide range of phenotypes, including an amyxomatous type, as well as the impact of invasive bacteria, that in part depended on the level of rabbit host resistance. These results provide a unique insight into the complex virus and host factors that combine to shape disease phenotype and viral evolution.

Evolutionary and genomic insights into the long-term colonization of Shigella flexneri in animals

ABSTRACT The enteroinvasive bacterium Shigella flexneri is known as a highly host-adapted human pathogen. There had been no known other reservoirs reported until recently. Here 34 isolates obtained from animals (yaks, dairy cows and beef cattle) from 2016 to 2017 and 268 human S. flexneri isolates from China were sequenced to determine the relationships between animal and human isolates and infer the evolutionary history of animal-associated S. flexneri. The 18 animal isolates (15 yak and 3 beef cattle isolates) in PG1 were separated into 4 lineages, and the 16 animal isolates (1 yak, 5 beef cattle and 10 dairy cow isolates) in PG3 were clustered in 8 lineages. The most recent human isolates from China belonged to PG3 whereas Chinese isolates from the 1950s–1960s belonged to PG1. PG1 S. flexneri may has been transmitted to the yaks during PG1 circulation in the human population in China and has remained in the yak population since, while PG3 S. flexneri in animals were likely recent transmissions from the human population. Increased stability of the large virulence plasmid and acquisition of abundant antimicrobial resistance determinants may have enabled PG3 to expand globally and replaced PG1 in China. Our study confirms that animals may act as a reservoir for S. flexneri. Genomic analysis revealed the evolutionary history of multiple S. flexneri lineages in animals and humans in China. However, further studies are required to determine the public health threat of S. flexneri from animals.

Genomic features of Mycoplasma bovis subtypes currently circulating in France

BackgroundMycoplasma (M.) bovis is a major etiological agent of bovine respiratory disease, which is the most economically costly disease of cattle worldwide. Cattle disease surveillance on M. bovis is increasingly using gene-based techniques, such as multilocus sequence typing (MLST), or genome-based techniques such as core genome MLST that both require only partial genomic data. However, accurate up-to-date surveillance also demands complete, circular genomes that can be used as reference to track the evolution of the different lineages. Yet, in France, two of the main subtypes currently circulating still have no representing genome in public databases. Here, to address this gap, we provide and compare three new complete M. bovis genomes obtained from recent clinical isolates that represent major subtypes circulating in France and Europe.ResultsGenomes were obtained using a hybrid assembly strategy (Illumina and Nanopore) with fine-tuning of settings and inputs used in the Unicycler assembly pipeline, such as size selection of reads and quality trimming of the FASTQ files. The main characteristics and synteny of the genomes were compared. The three genomes mainly differed by their content in terms of mobile genetic elements, i.e. integrative conjugative elements (ICE) and insertion sequences (IS), a feature that impacts their structure. For instance, strain L15527, representing subtype3 (st3), harbours an exceptionally high number of ICEs, which results in a bigger-sized genome than all those previously described and could be associated with the propensity of st3 to gain and fix mutations through chromosomal transfer mechanisms. In contrast, strain F9160, of st1, is very close to the PG45 type strain isolated in 1961 in the USA, and harbours a huge number of IS. These features may be associated with an evolution towards a host-restricted state or in a “closed” host or environment reservoir until a recent re-emergence.ConclusionsWhole-genome comparison of the three French M. bovis subtypes provides valuable resources for future studies combining epidemiology, phylogenetic data, and phylodynamic methods.

High genetic stability of co-circulating human adenovirus type 31 lineages over 59 years.

Type 31 of human adenovirus species A (HAdV-A31) is a significant pathogen primarily associated with diarrhoea in children but also with life-threatening disseminated disease in allogeneic haematopoietic stem cell transplant (HSCT) recipients. Nosocomial outbreaks of HAdV-A31 have been frequently described. However, the evolution of HAdV-A31 has not been studied in detail. The evolution of other HAdV types is driven either by intertypic recombination, where different types exchange genome regions, or by immune escape selection of neutralisation determinants. Complete genomic HAdV-A31 sequences from sixty diagnostic specimens of the past 18 years (2003-21) were generated, including fourteen specimens of a presumed outbreak on two HSCT wards. Additionally, twenty-three complete genomes from GenBank were added to our phylogenetic analysis as well as in silico generated and previously published restriction fragment polymorphism (RFLP) data. Phylogenetic analysis of eighty-three genomes indicated that HAdV-A31 evolved slowly with six lineages co-circulating. The two major lineages were lineage 1, which included the prototype from 1962 and nine recent isolates, and lineage 2, which split into four sublineages and included most isolates from 2003 to 2021. The average nucleotide identity within lineages was high (99.8 per cent) and identity between lineages was 98.7 and 99.2 per cent. RFLP data allowed the construction of a lower-resolution phylogeny with two additional putative lineages. Surprisingly, regions of higher diversity separating lineages were found in gene regions coding for non-structural and minor capsid proteins. Intertypic recombinations were not observed, but the phylogeny of lineage 3 was compatible with an interlineage recombination event in the fibre gene. Applying the phylogenetic analysis to the presumed nosocomial outbreak excluded two suspected transmission events and separated it into two different, simultaneous outbreaks caused by different sublineages of lineage 2. However, due to the high nucleotide identity within HAdV-A31 lineages, the proof of infection chains remains debatable. This in-depth study on the molecular phylogeny of HAdV-A31 highlights the high genetic stability of co-circulating HAdV-A31 lineages over almost six decades. It also supports the epidemiological hypothesis that HAdV-A31 circulates as an etiological agent of a childhood disease infecting immunologically naive patients without strong positive selection of immune escape variants and recombinants.

Candidate 'Avian orthoreovirus B': An emerging waterfowl pathogen in Europe and Asia?

A fusogenic virus was isolated from a flock of breeder Pekin ducks in 2019, Hungary. The affected flock experienced a marked decrease in egg production. Histopathological lesions were seen in the oviduct and in the lungs of birds sent for diagnostic investigation. The fusogenic agent was characterized as an orthoreovirus by viral metagenomics. The assembled viral genome was composed of 10 genomic segments and was 23,433 nucleotides (nt) in length. The study strain, designated Reo/HUN/DuckDV/2019, shared low-to-medium gene-wise sequence identity with avian orthoreovirus strains from galliform and anseriform birds (nt, 38.90%-72.33%) as well as with representative strains of neoavian orthoreoviruses (nt, 40.07%-68.23%). On the contrary, the study strain shared 86.48%-95.01% pairwise nt sequence identities with recent German and Chinese reovirus isolates, D2533/6 and Ych, respectively. Phylogenetic analysis clustered all three unusual waterfowl pathogens on a monophyletic branch, indicating a common evolutionary origin of Reo/HUN/DuckDV/2019 with these enigmatic orthoreoviruses described over the past few years. The finding that a candidate new orthoreovirus species, tentatively called Avian orthoreovirus B, was isolated in recent years in Europe and Asia in moribund ducks seems an alarming sign that needs to be better evaluated by extending laboratory diagnosis of viral pathogens in countries where the waterfowl industry is important.

The Development of 3D Bovine Intestinal Organoid Derived Models to Investigate Mycobacterium Avium ssp Paratuberculosis Pathogenesis.

Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of Johne's Disease, a chronic enteritis of ruminants prevalent across the world. It is estimated that approximately 50% of UK dairy herds are infected with MAP, but this is likely an underestimate of the true prevalence. Infection can result in reduced milk yield, infertility and premature culling of the animal, leading to significant losses to the farming economy and negatively affecting animal welfare. Understanding the initial interaction between MAP and the host is critical to develop improved diagnostic tools and novel vaccines. Here we describe the characterisation of three different multicellular in vitro models derived from bovine intestinal tissue, and their use for the study of cellular interactions with MAP. In addition to the previously described basal-out 3D bovine enteroids, we have established viable 2D monolayers and 3D apical-out organoids. The apical-out enteroids differ from previously described bovine enteroids as the apical surface is exposed on the exterior surface of the 3D structure, enabling study of host-pathogen interactions at the epithelial surface without the need for microinjection. We have characterised the cell types present in each model system using RT-qPCR to detect predicted cell type-specific gene expression, and confocal microscopy for cell type-specific protein expression. Each model contained the cells present in the original bovine intestinal tissue, confirming they were representative of the bovine gut. Exposure of the three model systems to the K10 reference strain of MAP K10, and a recent Scottish isolate referred to as C49, led to the observation of intracellular bacteria by confocal microscopy. Enumeration of the bacteria by quantification of genome copy number, indicated that K10 was less invasive than C49 at early time points in infection in all model systems. This study shows that bovine enteroid-based models are permissive to infection with MAP and that these models may be useful in investigating early stages of MAP pathogenesis in a physiologically relevant in vitro system, whilst reducing the use of animals in scientific research.Bos taurus: urn:lsid:zoobank.org:act:4C90C4FA-6296-4972-BE6A-5EF578677D64

Comparative study of the phenotype and virulence of recent serovar 1a, 1b, and 2a isolates of Erysipelothrix rhusiopathiae in Japan

Erysipelothrix rhusiopathiae causes swine erysipelas (SE) and is classified –into 16 serovars based on cell surface antigens. Our previous study suggested that recent SE outbreaks were mostly caused by serovar 1a of E. rhusiopathiae with the surface protective antigen (Spa)A protein characterized by methionine and isoleucine at positions 203 and 257 (M203/I257 SpaA). In this study, four recent E. rhusiopathiae isolates comprising two serovar 1a with M203/I257 SpaA strains (2012 Miyazaki and 2012 Chiba), one serovar 1b strain (2015 Miyazaki), and one serovar 2a strain (2012 Nagano) were compared with each other and with the serovar 1a Fujisawa reference strain regarding in vitro phenotypes and in vivo virulence in mice and pigs. The serovar 1b and 2a strains, which are the less prevalent strains in the field in Japan, showed lower growth in liquid culture and lower virulence in animals than the serovar 1a variants. Adhesion of the serovar 2a strain to porcine endothelial cells was weaker than that of the serovar 1a and 1b strains. Several advantages of serovar 1a strains were found, but no plausible cause of the M203/I257 SpaA type variants to be selected for the most prevalent strains among serovar 1a strains was identified in this study.

Combined knockdown of RL13 and UL128 for release of cell-free infectivity from recent HCMV isolates

Due to strictly cell-associated growth, experiments requiring cell-free virus are not applicable to recent clinical HCMV isolates to date. On the other hand, adaptation to cell-free growth is associated with undesirable changes in the viral gene regions RL13 and UL128. We had previously found that siRNA-mediated reduction of UL128 expression allowed transient release of cell-free virus by clinical isolates, and now hypothesized that virus yield could be further increased by additional knockdown of RL13. Despite the extensive polymorphism of RL13, effective RL13-specific siRNAs could be designed for three recent isolates and the Merlin strain. Knockdown efficiency was demonstrated at the protein level with a Merlin variant expressing V5-tagged pRL13. Knockdown of RL13 alone did not result in measurable release of cell-free virus, but combined knockdown of RL13 and UL128 increased infectivity in cell-free supernatants by a factor of 10–2000 compared to knockdown of UL128 alone. These supernatants could be used in dose-response assays to compare the effect of a neutralizing antibody on the various HCMV isolates. In summary, combined knockdown of RL13 and UL128 by specific siRNAs allows reliable release of cell-free infectivity from otherwise strictly cell-associated HCMV isolates without the need to modify the viral genome.

Activity of ceftolozane/tazobactam against Gram-negative isolates among different infections in Hong Kong: SMART 2017-2019.

Introduction. Ceftolozane/tazobactam was approved by the Drug Office, Department of Health, Government of the Hong Kong Special Administrative Region in 2017.Hypothesis/Gap Statement. Currently the in vitro activity of ceftolozane/tazobactam against Gram-negative pathogens isolated from patients in Hong Kong is undocumented. It would be prudent to document the activity of ceftolozane/tazobactam against Pseudomonas aeruginosa and Enterobacterales isolated from hospitalized patients in Hong Kong.Aim. To describe the in vitro susceptibility of recent clinical isolates of P. aeruginosa and the two most common Enterobacterales species (Klebsiella pneumoniae, Escherichia coli) cultured from respiratory tract, intra-abdominal, urinary tract and bloodstream infection samples to ceftolozane/tazobactam and other commonly used antimicrobial agents.Methodology. CLSI-defined broth microdilution MICs were determined and interpreted for Gram-negative isolates collected in Hong Kong from 2017 to 2019 by the SMART surveillance programme.Results. For P. aeruginosa, 96.7 % of isolates (n=210) were susceptible to ceftolozane/tazobactam, while susceptibility rates were ≥14 % lower to meropenem (82.9 % susceptible), cefepime (82.4 %), ceftazidime (81.4 %), piperacillin/tazobactam (76.7 %) and levofloxacin (79.5 %). Ceftolozane/tazobactam inhibited 85.7 % of piperacillin/tazobactam-nonsusceptible isolates, 80.6-82.1 % of cefepime-, ceftazidime- or meropenem-nonsusceptible isolates, and 75.9 % of multidrug-resistant (MDR) isolates of P. aeruginosa. For K. pneumoniae, 96.1 % of isolates (n=308) were susceptible to ceftolozane/tazobactam compared with meropenem (99.0 % susceptible), piperacillin/tazobactam (93.8 %), cefepime (85.7 %) and ceftazidime (85.4 %). The majority (88.3 %) of ESBL (extended-spectrum β-lactamase) non-CRE (carbapenem-resistant Enterobacterales) phenotype isolates of K. pneumoniae were susceptible to ceftolozane/tazobactam, comparable to piperacillin/tazobactam (85.0 %) but lower than meropenem (100 %). For E. coli, 98.5 % of isolates (n=609) were susceptible to ceftolozane/tazobactam compared to meropenem (99.3 % susceptible), piperacillin/tazobactam (96.7 %), ceftazidime (82.3 %) and cefepime (76.5 %). The majority (96.7 %) of ESBL non-CRE phenotype isolates of E. coli were susceptible to ceftolozane/tazobactam, similar to both meropenem (100 %) and piperacillin/tazobactam (94.5 %).Conclusions. Overall, >96 % of clinical isolates of P. aeruginosa, K. pneumoniae and E. coli collected in Hong Kong in 2017-2019 were susceptible to ceftolozane/tazobactam, while the activity of several commonly prescribed β-lactams was reduced, especially for P. aeruginosa. Continued surveillance of ceftolozane/tazobactam and other agents is warranted.

Antimicrobial susceptibility testing of clinical isolates of Gram-negative bacilli collected in Morocco by the ATLAS Global Surveillance Program from 2018 to 2020

To report reference method antimicrobial susceptibility testing results for recent clinical isolates of Gram-negative bacilli from Morocco. CLSI (Clinical and Laboratory Standards Institute) broth microdilution antimicrobial susceptibility testing was performed by a central laboratory for isolates of Enterobacterales (n=810), Pseudomonas aeruginosa (n=321), and Acinetobacter baumannii (n=191) collected in 2018-2020 by three hospital laboratories in Morocco. MICs were interpreted using both CLSI (2021) and EUCAST (European Committee on Antimicrobial Susceptibility Testing) (2021) breakpoints. Molecular testing for β-lactamase genes was performed on isolates meeting defined screening criteria. Most isolates of Enterobacterales were susceptible (CLSI/EUCAST breakpoints) to amikacin (98.0%/96.2%), ceftazidime-avibactam (94.8%/94.8%), and meropenem (92.5%/94.2%). Of Enterobacterales isolates eligible for β-lactamase gene screening (n=210), 174 were ESBL-positive, 40 were metallo-β-lactamase-positive (all NDM), 39 were serine carbapenemase-positive (all OXA); and 7 isolates carried both OXA-48 and NDM-1. Amikacin (89.1%/89.1%) and ceftazidime-avibactam (88.2%/88.2%) were the most active agents tested against P. aeruginosa. Applying CLSI and EUCAST breakpoints, MDR rates were 21.9% and 29.3% for Enterobacterales and 18.4% and 21.8% for P. aeruginosa. Susceptible rates for amikacin, ceftazidime-avibactam, and meropenem were 93.2%/89.5%, 77.4%/82.3%, and 67.8%/80.2% for MDR Enterobacterales and 50.8%/57.1%, 40.7%/45.7%, and 27.1/32.9% for MDR P. aeruginosa. ≥70% of A. baumannii isolates were resistant to all agents tested (except colistin, EUCAST breakpoints only) including amikacin and meropenem. Newer β-lactam/β-lactamase inhibitor combinations such as ceftazidime-avibactam warrant testing and reporting for Enterobacterales and P. aeruginosa in Morocco given the presence of significant resistance to first-line β-lactams and fluoroquinolones, pervasive ESBLs and carbapenemases, and toxicity concerns associated with some second-line agents.

Pathogenesis of recent Lassa virus isolates from lineages II and VII in cynomolgus monkeys

ABSTRACT The area of Lassa virus (LASV) circulation is expanding, with the emergence of highly pathogenic new LASV lineages. Benin recently became an endemic country for LASV and has seen the emergence of a new LASV lineage (VII). The first two outbreaks in 2014 and 2016 showed a relatively high mortality rate compared to other outbreaks. We infected cynomolgus monkeys with two strains belonging to lineage II and lineage VII that were isolated from deceased patients during the 2016 outbreak in Benin. The lineage VII strain (L7) caused uniform mortality. Death was associated with uncontrolled viral replication, unbalanced inflammatory responses characterized by increased concentrations of pro- and anti-inflammatory mediators, and the absence of efficient immune responses, resembling the pathogenesis associated with the prototypic Josiah strain in monkeys. The lineage II strain (L2) showed apparently lower virulence than its counterpart, with a prolonged time to death and a lower mortality rate. Prolonged survival was associated with better control of viral replication, a moderate inflammatory response, and efficient T-cell responses. Transcriptomic analyses also highlighted important differences in the immune responses associated with the outcome. Both strains caused strong inflammation in several organs. Notably, meningitis and encephalitis were observed in the cerebral cortex and cerebellum in all monkeys, independently of the outcome. Due to their apparently high pathogenicity, emerging strains from lineage VII should be considered in preclinical vaccine testing. Lineage II would also be beneficial in pathogenesis studies to study the entire spectrum of Lassa fever severity.

A Zika virus mutation enhances transmission potential and confers escape from protective dengue virus immunity.

SUMMARYZika virus (ZIKV) and dengue virus (DENV) are arthropod-borne pathogenic flaviviruses that co-circulate in many countries. To understand some of the pressures that influence ZIKV evolution, we mimic the natural transmission cycle by repeating serial passaging of ZIKV through cultured mosquito cells and either DENV-naive or DENV-immune mice. Compared with wild-type ZIKV, the strains passaged under both conditions exhibit increased pathogenesis in DENV-immune mice. Application of reverse genetics identifies an isoleucine-to-valine mutation (I39V) in the NS2B proteins of both passaged strains that confers enhanced fitness and escape from pre-existing DENV immunity. Introduction of I39V or I39T, a naturally occurring homologous mutation detected in recent ZIKV isolates, increases the replication of wild-type ZIKV in human neuronal precursor cells and laboratory-raised mosquitoes. Our data indicate that ZIKV strains with enhanced transmissibility and pathogenicity can emerge in DENV-naive or -immune settings, and that NS2B-I39 mutants may represent ZIKV variants of interest.

Evaluation of in vitro activity of ceftolozane/tazobactam and comparators against recent clinical bacterial isolates, and genomics of Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli isolates that demonstrated resistance to ceftolozane/tazobactam: data from Kuwait and Oman.

BackgroundThe treatment options for infections caused by MDR Gram-negative bacteria have been limited, especially for infections caused by bacteria that produce carbapenemases and/or ESBLs. Ceftolozane/tazobactam is a cephalosporin/β-lactamase inhibitor developed to treat Gram-negative bacteria.MethodsCeftolozane/tazobactam and 14 comparators (amikacin, aztreonam, cefepime, cefotaxime, cefoxitin, ceftazidime, ceftriaxone, ciprofloxacin, colistin, ertapenem, imipenem, levofloxacin, meropenem and piperacillin/tazobactam) were evaluated against Pseudomonas aeruginosa and Enterobacterales isolates collected from Kuwait and Oman (n = 606) during 2016–17. In addition, further analysis of resistance mechanisms to ceftolozane/tazobactam was done utilizing WGS. Non-susceptible isolates from ceftolozane/tazobactam surveillance were selected for analysis. Overall, 35 strains underwent WGS.ResultsAmong isolates from Kuwait, susceptibility of P. aeruginosa, Escherichia coli and Klebsiella pneumoniae to ceftolozane/tazobactam was 79.8%, 95.7% and 87.5%, respectively, and from Oman was 92.3%, 93.1% and 88.5%, respectively. No P. aeruginosa with a ceftolozane/tazobactam MIC <32 mg/L encoded β-lactamases besides normal chromosomal enzymes (PDC variants or OXA-50-like) whereas all but one P. aeruginosa isolate with MIC >32 mg/L encoded either MBLs (60%), VEB-1 (19%) or additional OXAs (3.7%).ConclusionsColistin followed by ceftolozane/tazobactam showed the greatest activity against P. aeruginosa. Enterobacterales showed more susceptibility to ceftolozane/tazobactam than to piperacillin/tazobactam, but meropenem and colistin showed better activity.

Rapid and Accurate Differentiation of Mycobacteroides abscessus Complex Species by Liquid Chromatography-Ultra-High-Resolution Orbitrap™ Mass Spectrometry.

In this study, a Liquid Chromatography-Mass Spectrometry (LC-MS) method for the identification of clinically relevant Mycobacteroides abscessus (Mabs) complex organisms is tested using a set of microbial Type strains. This methodology is based on profiling proteins derived from Mycobacteroides abscessus complex isolates. These protein profiles are then used as markers of species differentiation. To test the resolving power, speed, and accuracy of this assay four ATCC type strains and 32 recent clinical isolates of closely related Mabs species collected at ARUP laboratories (10 clinical isolate strains of M. abscessus subsp. abscessus, 10 M. abscessus subsp. massiliense, 2 M. abscessus subsp. bolletii and 10 M. chelonae) were subjected to this approach. Using multiple deconvolution algorithms, we identified hundreds of individual proteins, with subpopulations of these used as species-specific markers. This assay identified 150, 130, 140 and 110 proteoforms with isocratic elution and 230, 180, 200 and 190 proteoforms with gradient elution for M. abscessus (ATCC 19977), M. massiliense (DSM 45103), M. bolletii (DSM 45149) and M. chelonae (ATCC 35752) respectively. Taxonomic species were identified correctly down to the species level with 100% accuracy. The ability to differentiate Mycobacteroides abscessus complex at sub-species level can in-turn be helpful for patient management. Data analysis showed ~7-17 proteoforms potentially able to differentiate between subspecies. Here, we present a proof-of-principle study employing a rapid mass spectrometry-based method to identify the clinically most common species within the Mabs species complex.

Construction of full-length infectious clones of turnip mosaic virus isolates infecting Perilla frutescens and genetic analysis of recently emerged strains in Korea

Perilla is an annual herb with a unique aroma and taste that has been cultivated in Korea for hundreds of years. It has been widely cultivated in many Asian and European countries as a food and medicinal crop. Recently, several viruses have been reported to cause diseases in perilla in Korea, including turnip mosaic virus (TuMV), which is known as a brassica pathogen due to its significant damage to brassica crops. In this study, we determined the complete genome sequences of two new TuMV isolates originating from perilla in Korea. Full-length infectious cDNA clones of these two isolates were constructed, and their infectivity was tested by agroinfiltration of Nicotiana benthamiana and sap inoculation of Chinese cabbage and radish plants. In addition, we analyzed the phylogenetic relationship of six new Korean TuMV isolates to members of the four major groups. We also used RDP4 software to conduct recombination analysis of recent isolates from Korea, which provided new insight into the evolutionary relationships of Korean isolates of TuMV.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00705-021-05356-9.

Staphylococcus aureus in Non-Cystic Fibrosis Bronchiectasis: Prevalence and Genomic Basis of High Inoculum β-Lactam Resistance.

Rationale: The pathobiology of Staphylococcus aureus in non-cystic fibrosis bronchiectasis (nCFB) is poorly defined. When present at high density or "inoculum," some methicillin-sensitive S. aureus (MSSA) can inefficiently degrade antistaphylococcal β-lactam antibiotics via BlaZ penicillinases (termed the "inoculum effect" [IE]). Given the high burden of organisms in bronchiectatic airways, this is particularly relevant. Objectives: Drawing from a prospectively collected biobank, we sought to understand the prevalence, natural history, potential for transmission, and antibiotic resistance profiles among nCFB-derived MSSA isolates. Methods: All individuals attending a regional consultancy nCFB clinic with sputum collected between 1981 and 2017 were considered, and those with one or more S. aureus-positive cultures composed the cohort. Each individual's most recent biobank isolate was subjected to whole-genome sequencing (including the blaZ gene), antibacterial susceptibility testing, and comparative β-lactam testing at standard (5 × 105 colony-forming unit [cfu]/ml) and high (5 × 107 cfu/ml) inocula to assess for the IE and pronounced IE. Results: Seventy-four (35.4%) of 209 individuals had one or more sputum samples with S. aureus (68 MSSA, 6 methicillin-resistant S. aureus). Those with S. aureus infection were more likely to be female. Among 60 of 74 MSSA isolates subjected to whole-genome sequencing, no evidence of transmission was identified, although specific multilocus sequence typing types were prevalent, including ST-1, ST-15, ST-30, and ST-45. Antibiotic resistance was uncommon, except for macrolides (∼20%). Among the 60 MSSA samples, the prevalence of IE and pronounced IE was observed to be drug specific: meropenem (0% and 0%, respectively), cefepime (3% and 5%, respectively), ceftazidime (8% and 0%, respectively), cloxacillin (12% and 0%, respectively), cefazolin (23% and 0%, respectively), and piperacillin-tazobactam (37% and 17%, respectively). The cefazolin IE was associated with blaZ type A (P < 0.01) and ST-30 (P < 0.01), whereas the piperacillin-tazobactam IE was associated with type C blaZ (P < 0.001) and ST-15 (P < 0.05). Conclusions:S. aureus infection was common, although no evidence of transmission was apparent in our nCFB cohort. Although routine susceptibility testing did not identify significant resistance, inoculum-related resistance was found to be relevant for commonly used nCFB antibiotics, including cefazolin and piperacillin-tazobactam. Given previous associations between IEs and negative patient outcomes, further work is warranted to understand how this phenotype impacts nCFB disease progression.

Bbvac: A Live Vaccine Candidate That Provides Long-Lasting Anamnestic and Th17-Mediated Immunity against the Three Classical Bordetella spp.

ABSTRACTAcute pathogens such as Bordetella pertussis can cause severe disease but are ultimately cleared by the immune response. This has led to the accepted paradigm that convalescent immunity is optimal and therefore broadly accepted as the “gold standard” against which vaccine candidates should be compared. However, successful pathogens like B. pertussis have evolved multiple mechanisms for suppressing and evading host immunity, raising the possibility that disruption of these mechanisms could result in substantially stronger or better immunity. Current acellular B. pertussis vaccines, delivered in a 5-dose regimen, induce only short-term immunity against disease and even less against colonization and transmission. Importantly, they provide modest protection against other Bordetella species that cause substantial human disease. A universal vaccine that protects against the three classical Bordetella spp. could decrease the burden of whooping cough-like disease in humans and other animals. Our recent work demonstrated that Bordetella spp. suppress host inflammatory responses and that disrupting the regulation of immunosuppressive mechanisms can allow the host to generate substantially stronger sterilizing immunity against the three classical Bordetella spp. Here, we identify immune parameters impacted by Bordetella species immunomodulation, including the generation of robust Th17 and B cell memory responses. Disrupting immunomodulation augmented the immune response, providing strong protection against the prototypes of all three classical Bordetella spp. as well as recent clinical isolates. Importantly, the protection in mice lasted for at least 15 months and was associated with recruitment of high numbers of B and T cells in the lungs as well as enhanced Th17 mucosal responses and persistently high titers of antibodies. These findings demonstrate that disrupting bacterial immunomodulatory pathways can generate much stronger and more protective immune responses to infection, with important implications for the development of better vaccines.IMPORTANCE Infectious diseases are a major cause of morbidity and mortality in the United States, accounting for over 40 million hospitalizations since 1998. Therefore, novel vaccine strategies are imperative, which can be improved with a better understanding of the mechanisms that bacteria utilize to suppress host immunity, a key mechanism for establishing colonization. Bordetella spp., the causative agents of whooping cough, suppress host immunity, which allows for persistent colonization. We discovered a regulator of a bacterial immunosuppressive pathway, which, when mutated in Bordetella spp., allows for rapid clearance of infection and subsequent generation of protective immunity for at least 15 months. After infection with the mutant strain, mice exhibited sterilizing immunity against the three classical Bordetella spp., suggesting that the immune response can be both stronger and cross-protective. This work presents a strategy for vaccine development that can be applied to other immunomodulatory pathogens.

In-vitro activity of ceftolozane/tazobactam against recent clinical bacterial isolates from two Saudi Arabian hospitals

BackgroundAntimicrobial resistance is continuously increasing. Adding tazobactam to ceftolozane improves the latter’s activity spectrum against resistant strains. We aimed to determine the susceptibility of recently collected bacterial isolates to ceftolozane/tazobactam (C/T) and other antibiotics. MethodsThis was a prospective cohort study conducted between March 2017 and March 2018. The in-vitro activities of C/T and 14 other antibiotics were assessed against 192 gram-negative bacterial (GNB) isolates (P. aeruginosa, K. pneumonia, E. coli, and other Enterobacterales) prospectively collected from two hospitals in Saudi arabia; in the laboratories of the International Health Management Associates Inc. Samples were obtained from intensive care units (ICUs) and non-ICU locations. The minimum inhibitory concentrations (MICs) of the antibiotics were determined by broth microdilution. Isolates were obtained from different infection sites [urine (31.8%), urinary bladder samples (15.1%), abscess/pus (20.3%), endotracheal aspirates (18.8%)]. ResultsOur sample showed substantial drug resistance; 66.1% of the collected isolates showed either multiple or extensive drug resistance. Susceptibility rates of P. aeruginosa (n = 50), E.coli (n = 40), K. pneumoniae (n = 64) and other Enterobacterales (n = 38) to C/T were 74%, 87.5%, 48.4% and 71.1%, respectively. According to MIC50 values (1 µg/mL for both P. aeruginosa and other Enterobacterales, 0.5 µg/mL for E.coli, and 4 µg/mL for K. pneumoniae), C/T was among the most potent antibiotics against these isolates. ConclusionsC/T displayed high potency against all examined bacterial isolates. It was mainly active against E.coli followed by P.aeruginosa and other Enterobacterales and its lowest susceptibility rate was reported against K. pneumoniae.

P43 In vitro activity of cefiderocol and comparators against Gram-negative pathogens: ARTEMIS study in the UK

ObjectivesCefiderocol (CFDC) is a novel siderophore cephalosporin approved in Europe for the treatment of infections caused by aerobic Gram-negative (GN) bacteria in adults with limited treatment options. The aim of the ARTEMIS study was to evaluate the in vitro activity of CFDC and comparators against recent clinical isolates collected across five countries in Europe. Here we report susceptibility data from isolates collected in the UK.MethodsFrom January to December 2020, GN clinical isolates were collected from hospitalized patients from all infection sites (excluding the urinary tract). Duplicate isolates of the same species from a single patient were excluded. As a prespecified target, each laboratory collected 75 isolates, with: 20 Klebsiella spp., 20 other Enterobacterales, 20 Pseudomonas aeruginosa and 15 Acinetobacter baumannii isolates expected to be included. CFDC susceptibility testing was conducted using disc diffusion (with 30 μg discs) on Mueller–Hinton agar and Sensititre™ broth microdilution (BMD) panels [EUMDROXF; centrally tested at International Health Management Associates (IHMA)]. Susceptibility by disc diffusion was reported using zone diameter breakpoints (BPs) of ≥22 mm (or ≥17 mm for A. baumannii isolates, corresponding to MIC values below the pharmacokinetic/pharmacodynamic BPs of ≤2 mg/L). Comparator susceptibility was determined using custom research use only Sensititre™ BMD panels (CMP2SHIH) according to the EUCAST method for BMD. Antimicrobial susceptibility was interpreted according to EUCAST clinical BPs (v.11 2021).ResultsIn total, 517 isolates were collected from nine UK hospitals, of which: 308 (59.6%) were Enterobacterales [including 147 (28.4%) Klebsiella spp.], 148 (28.6%) were P. aeruginosa and 33 (6.4%) were A. baumannii. The most common sites of infection were bloodstream (n = 245; 47.4%), respiratory tract (n = 158; 30.6%) and skin (n = 59; 11.4%). A high percentage of Enterobacterales (90.2%), P. aeruginosa (96.6%) and A. baumannii (96.9%) isolates were susceptible to CFDC by disc diffusion. By central laboratory testing (MIC), 99.0% of Enterobacterales, 99.3% of P. aeruginosa and 93.9% of A. baumannii isolates were susceptible to CFDC. High susceptibility rates (>85%) were also observed for all comparator agents (Table 1). A total of 32/517 (6.2%) isolates were carbapenem resistant, the majority of which (22/32, 68.8%) were susceptible to CFDC by disc diffusion.Table 1.CFDC susceptibility based on disc zone diameters; CFDC and comparator agent susceptibility based on MIC from BMD (EUCAST clinical BPs v.11, 2021)Susceptibility, n/N (%)CFDC (disc)No. isolates in ATUCFDC (MIC)MEMaC/TCZAMVBI/RCSTEnterobacterales277/307 (90.2)40/307 (13.0)285/288 (99.0)295/308 (95.8)264/308 (85.7)298/307 (97.1)303/308 (98.4)286/306 (93.5)265/307 (86.3) P. aeruginosa 143/148 (96.6) 8/148 (5.4)144/145 (99.3)135/148 (91.2)140/148 (94.6)141/148 (95.3)137/148 (92.6)133/147 (90.5)142/148 (95.9) A. baumannii 31/32 (96.9)N/A31/33 (93.9)30/33 (90.9)N/AN/AN/A30/33 (90.9)30/33 (90.9)ATU, area of technical uncertainty; CFDC, cefiderocol; CST, colistin; C/T, ceftolozane/tazobactam; CZA, ceftazidime/avibactam; I/R, imipenem/relebactam; MEM, meropenem; MVB, meropenem/vaborbactam.aIsolates were defined as MEM-susceptible with a BP of ≤8 mg/L [relating to high-dose extended-infusion (2 g, 3 h infusion) MEM], based on EUCAST recommendations.ConclusionsAmong clinical GN isolates collected from UK hospitals in 2020, a high percentage (98.6%), including carbapenem-resistant isolates, were susceptible to CFDC by BMD. These data support the use of CFDC in patients with GN infections and limited treatment options. The differences identified between EUCAST disc diffusion and BMD using Sensititre™ panels for CFDC highlight that disc diffusion underestimates Enterobacterales susceptibility to CFDC, which is mainly a result of the area of technical uncertainty (where isolates with MIC of 2 mg/L have a zone diameter of <22 mm and are characterized as resistant). This requires further investigation to explore whether the EUCAST zone diameter BP is optimal for CFDC disc testing.