Recent Isolates Research Articles

597. Cross-resistance of Ceftolozane-Tazobactam and Imipenem-Relebactam Against Clinical P. aeruginosa Isolates: SMART United States 2016–2018

BackgroundCeftolozane-tazobactam (C/T) is an antipseudomonal cephalosporin combined with a β-lactamase inhibitor. The combination was cleared by FDA and EMA and is approved in the United States and over 60 countries worldwide. Relebactam (REL) is an inhibitor of class A and C β-lactamases that is in clinical development in combination with imipenem (IMI). Using clinical isolates collected in the United States as part of the global SMART surveillance program, we compared the activity of C/T and IMI/REL against P. aeruginosa (PA) isolates.MethodsIn 2016–2018, 29 clinical laboratories from the United States collected up to 250 consecutive, aerobic or facultatively anaerobic, gram-negative pathogens (GNP) from blood, intra-abdominal, urinary, and lower respiratory tract infections. A total of 14,606 GNP were collected, of which 2,774 were PA. MICs were determined using CLSI broth microdilution and interpreted with CLSI 2019 breakpoints; IMI breakpoints were used for IMI/REL.ResultsThe activity of C/T and IMI/REL against 2,774 PA is shown (table). Among all PA, 1.8% of isolates were nonsusceptible (NS) to both agents; 4.4% were susceptible (S) to C/T but not to IMI/REL, and 2.9% were susceptible to IMI/REL but not to C/T. Among the subset of isolates collected from patients in ICUs (n = 827), 87.3% were susceptible to both C/T and IMI/REL, 2.7% were nonsusceptible to both agents, 5.8% of isolates were susceptible only to C/T, and 4.2% of isolates were susceptible only to IMI/REL. Among all C/T-NS isolates (all patient locations, n = 132), 61.4% were IMI/REL-S and <30% were susceptible to all other studied β-lactams and fluoroquinolones. Among all IMI/REL-NS isolates (n = 173), 70.5% were C/T-S and <36% were susceptible to all other studied β-lactams and fluoroquinolones. Of the tested agents, only amikacin and colistin exceeded the activity of C/T or IMI/REL against these NS subsets.ConclusionResistance to C/T or IMI/REL was not common among recent clinical isolates of PA collected in the United States, and both agents promise to be important treatment options. A significant proportion of isolates nonsusceptible to one agent was susceptible to the other, especially among isolates from patients in ICUs. The data suggest that susceptibility to both agents should be tested at hospitals. Disclosures All authors: No reported disclosures.

Neurotropism of Enterovirus D68 Isolates Is Independent of Sialic Acid and Is Not a Recently Acquired Phenotype.

Acute flaccid myelitis (AFM) is a rare but serious illness of the nervous system, specifically affecting the gray matter of the spinal cord, motor-controlling regions of the brain, and cranial nerves. Most cases of AFM are pathogen associated, typically with poliovirus and enterovirus infections, and occur in children under the age of 6 years. Enterovirus D68 (EV-D68) was first isolated from children with pneumonia in 1962, but an association with AFM was not observed until the 2014 outbreak. Organotypic mouse brain slice cultures generated from postnatal day 1 to 10 mice and adult ifnar knockout mice were used to determine if neurotropism of EV-D68 is shared among virus isolates. All isolates replicated in organotypic mouse brain slice cultures, and three isolates replicated in primary murine astrocyte cultures. All four EV-D68 isolates examined caused paralysis and death in adult ifnar knockout mice. In contrast, no viral disease was observed after intracranial inoculation of wild-type mice. Six of the seven EV-D68 isolates, including two from 1962 and four from the 2014 outbreak, replicated in induced human neurons, and all of the isolates replicated in induced human astrocytes. Furthermore, a putative viral receptor, sialic acid, is not required for neurotropism of EV-D68, as viruses replicated within neurons and astrocytes independent of binding to sialic acid. These observations demonstrate that EV-D68 is neurotropic independent of its genetic lineage and can infect both neurons and astrocytes and that neurotropism is not a recently acquired characteristic as has been suggested. Furthermore, the results show that in mice the innate immune response is critical for restricting EV-D68 disease.IMPORTANCE Since 2014, numerous outbreaks of childhood infections with enterovirus D68 (EV-D68) have occurred worldwide. Most infections are associated with flu-like symptoms, but paralysis may develop in young children. It has been suggested that infection only with recent viral isolates can cause paralysis. To address the hypothesis that EV-D68 has recently acquired neurotropism, murine organotypic brain slice cultures, induced human motor neurons and astrocytes, and mice lacking the alpha/beta interferon receptor were infected with multiple virus isolates. All EV-D68 isolates, from 1962 to the present, can infect neural cells, astrocytes, and neurons. Furthermore, our results show that sialic acid binding does not play a role in EV-D68 neuropathogenesis. The study of EV-D68 infection in organotypic brain slice cultures, induced motor neurons, and astrocytes will allow for the elucidation of the mechanism by which the virus infection causes disease.

Reduced Virulence of an Introduced Forest Pathogen over 50 Years.

Pathogen incursions are a major impediment for global forest health. How pathogens and forest trees coexist over time, without pathogens simply killing their long-lived hosts, is a critical but unanswered question. The Dothistroma Needle Blight pathogen Dothistroma septosporum was introduced into New Zealand in the 1960s and remains a low-diversity, asexual population, providing a unique opportunity to analyze the evolution of a forest pathogen. Isolates of D. septosporum collected from commercial pine forests over 50 years were compared at whole-genome and phenotype levels. Limited genome diversity and increased diversification among recent isolates support the premise of a single introduction event. Isolates from the 1960s show significantly elevated virulence against Pinus radiata seedlings and produce higher levels of the virulence factor dothistromin compared to isolates collected in the 1990s and 2000s. However, later isolates have no increased tolerance to copper, used in fungicide treatments of infested forests and traditionally assumed to be a strong selection pressure. The isolated New Zealand population of this forest pathogen therefore appears to have become less virulent over time, likely in part to maintain the viability of its long-lived host. This finding has broad implications for forest health and highlights the benefits of long-term pathogen surveys.

Effector gene reshuffling involves dispensable mini-chromosomes in the wheat blast fungus.

Newly emerged wheat blast disease is a serious threat to global wheat production. Wheat blast is caused by a distinct, exceptionally diverse lineage of the fungus causing rice blast disease. Through sequencing a recent field isolate, we report a reference genome that includes seven core chromosomes and mini-chromosome sequences that harbor effector genes normally found on ends of core chromosomes in other strains. No mini-chromosomes were observed in an early field strain, and at least two from another isolate each contain different effector genes and core chromosome end sequences. The mini-chromosome is enriched in transposons occurring most frequently at core chromosome ends. Additionally, transposons in mini-chromosomes lack the characteristic signature for inactivation by repeat-induced point (RIP) mutation genome defenses. Our results, collectively, indicate that dispensable mini-chromosomes and core chromosomes undergo divergent evolutionary trajectories, and mini-chromosomes and core chromosome ends are coupled as a mobile, fast-evolving effector compartment in the wheat pathogen genome.

Genotypic evolution and epidemiological characteristics of H9N2 influenza virus in Shandong Province, China.

H9N2 avian influenza has been prevalent in chicken flocks of China for years. In the first half year of 2018, clinical cases of suspected H9N2 infection were collected from chicken flocks in Shandong province. Nine strains of H9N2 influenza virus were isolated. The pathological changes of the dead chickens were mainly respiratory inflammation, renal swelling, and secondary infection. The microscopic lesions were consistent with the pathogenic characteristics of H9N2 influenza virus. From November 2017 to June 2018, a total of 3,380 serum samples were randomly collected from commercial laying hens in Shandong Province. The H9 antibody levels were tested with the isolated strain (CK/SD/231/17) as the antigen. It showed that the average of antibody titers of H9 avian influenza was 9.24 1og2. Hemagglutination inhibition experiments were conducted on chicken serum with the vaccine virus and the isolated virus (CK/SD/231/17) as the antigens. It was found that the antibody titer measured with the vaccine virus was 1 or 2 titers higher than the isolated strain. It indicated that the antigenicity of H9N2 circulating strain was different from that of vaccine strain. The nucleotide sequences of HA gene of these recent H9N2 avian influenza virus isolates shared homologies from 93.8 to 99.9%. Phylogenetic analysis revealed that the eight gene segments of the viruses were in the same clades with G57 gene reference strain. The amino acid site analysis of influenza resistance showed that the virus was sensitive to neuraminidase inhibitors and resistant to amantadine. Highlights: The protection rate of the H9N2 AIV vaccine almost reached 100% before 2016, but the antibody level of serum samples showed high diversity in this study, which means the poultry were infected. The antigenicity of isolated H9N2 strains was different from that of vaccine strain. Current available vaccines may provide only limited protection.

CP-CRE/non-CP-CRE Stratification And CRE Resistance Mechanism Determination Help In Better Managing CRE Bacteremia Using Ceftazidime-Avibactam And Aztreonam-Avibactam.

PurposeThis observational study aimed to identify the independent risk factors for both the acquisition and mortality of carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) bacteremia and further assess the in vitro antimicrobial activities of ceftazidime–avibactam (CAZ/AVI) and aztreonam–avibactam (ATM/AVI) against recent CRE bacteremic isolates.Patients and methodsThis observational study was conducted to reveal the risk factors and mortality rate for CP-CRE bacteremia between 2012 and 2018 and also evaluate the in vitro antimicrobial activities of CAZ/AVI and ATM/AVI against recent CRE bacteremic isolates from 2016 to 2018.ResultsA total of 81 non-repetitive isolates were collected from 2012 to 2018, with 67.90% (55/81) being CP-CRE. Old age (P = 0.01), transfusion [odds ratio (OR): 17.19; 95% CI: 3.15–93.72; P = 0.001], longer ICU stay (P = 0.02), cancer (OR: 15.91; 95% CI: 3.56–71.37; P < 0.001), and previous carbapenem exposure (OR: 27.86; 95% CI: 5.03–154.19; P = 0.001) were identified as independent risk factors for the acquisition of CP-CRE bacteremia compared with the ESBL bacteremia. The in vitro antimicrobial activities of CAZ/AVI and ATM/AVI against the CRE bacteremic isolates from 2016 to 2018 showed a respective susceptibility rate of 70.68% (41/58) and 100.00% (58/58).ConclusionThe findings indicated that both CP-CRE/non-CP-CRE stratification and CRE resistance mechanism determination were necessary for better guiding the clinical management of CRE bacteremia: ATM/AVI probably works with both non-CP-CRE and CP-CRE bacteremia, even the most notorious double-carbapenemase producer with porin loss/deficiency, whereas CAZ/AVI works with most of the non-CP-CRE and KPC-producers in the region.

Authenticating Anaerobes – Use of MALDI-TOF MS to identify anaerobes in the NCTC Collection

The National Collection of Type Cultures (NCTC) is the world’s oldest repository of medically-relevant bacteria. NCTC contains 5,500 bacterial strains, 500 of which are anaerobes, with the collection regularly receiving new Type strains and recent clinical isolates. Fastidious anaerobes pose a unique challenge during the authentication process. NCTC must ensure that the strains are free from contamination and that the organism survives the lyophilisation process. Species-level identification of anaerobic bacterial strains is achieved using a combination of the both MALDI-TOF MS and VITEK2 instruments. This study evaluates the use of MALDI-TOF MS (Bruker) and VITEK2 (BioMerieux) to identify the anaerobic strains in the NCTC collection. 176 NCTC strains were tested on the MALDI-TOF platform and 60 strains were identified using VITEK2. MALDI-TOF was able to identify 79% anaerobes to genus-level and 64% to species-level. In comparison the VITEK2 identified 68% to genus and 46% to species-level. The main limiting factor for both these platforms is the database. This may be due to novel anaerobe species NCTC receives not being present on the databases. In the event of no identification, 16S ribosomal RNA sequencing is employed. Furthermore, detection of specific characteristics is carried out by specialist reference laboratories in Cardiff and Colindale, ensuring a robust collection of anaerobic bacteria for use in research and as control strains.

Molecular genomic characterization of severe fever with thrombocytopenia syndrome virus isolates from South Korea

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne emerging infectious disease caused by the SFTS virus (SFTSV) and is a threat to public health due to its high fatality rate. However, details on tick-to-human transmission of SFTSV are limited. In this study, we determined the whole-genome sequence of a South Korean SFTSV strain (CUK-JJ01), compared it to those of other recent human SFTSV isolates, and identified the genetic variations and relationships among the SFTSV strains. The genome of CUK-JJ01 was consistent with the genome of other members of the genus Phlebovirus, including the large (L), medium (M), and small (S) segments of 6368, 3378, and 1744 nucleotides, respectively. Based on amino acid sequences of the M and S segments, which are used to distinguish the six SFTSV genotypes, CUK-JJ01 was classified as genotype B. Segment analysis revealed that the L, M, and S segments were 97.49%, 97.18%, and 97.94% similar to those of KAJNH2/2013/Korea, ZJZHSH-FDE/2012/China, and KADGH/2013/Korea, respectively. Currently, only few studies on SFTSV have been conducted in Korean population and most were limited to serological analysis. Although the present study has limitations in terms of number of sample analyzed, the findings may serve as basis to understand the transmission and spread of SFTSV, as well as for the development of diagnostic and detection methods for viral recombinants by comparing the whole genome sequence of SFTSV isolates from South Korea and that of foreign isolates.

In vitro activity of Exebacase (CF-301) against clinical Staphylococcus aureus surveillance isolates from the United States, Europe, and Latin America, 2015–2017

Lysins are direct lytic agents which act through enzymatic cell-wall-hydrolysis and represent a potential new class of antimicrobial agents in development to treat antibiotic-resistant bacterial infections. Exebacase (CF-301) is a first-in-class lysin now in clinical development for the treatment of Staphylococcus aureus (S. aureus) bacteremia and infective endocarditis (IE) when used in addition to conventional antibiotics. Exebacase and comparator antibiotics were tested by broth microdilution against a set of 535 clinical MSSA and MRSA isolates collected from 2015 to 2017 throughout the United States, Europe and South America. All S. aureus isolates were inhibited by ≤1 mg/L exebacase (MIC50/90, 0.5/1 mg/L) with a range of 0.25–1 mg/L. No difference in susceptibility was observed between the MSSA and MRSA isolates. Exebacase was uniformly and equivalently active against all recent clinical MSSA and MRSA surveillance isolates from a broad survey across 3 continents.

In Vitro Activity of APX001A (Manogepix) and Comparator Agents against 1,706 Fungal Isolates Collected during an International Surveillance Program in 2017.

Current antifungal agents cover a majority of opportunistic fungal pathogens; however, breakthrough invasive fungal infections continue to occur and increasingly involve relatively uncommon yeasts and molds, which often exhibit decreased susceptibility. APX001A (manogepix) is a first-in-class small-molecule inhibitor of the conserved fungal Gwt1 protein. This enzyme is required for acylation of inositol during glycosylphosphatidylinositol anchor biosynthesis. APX001A is active against the major fungal pathogens, i.e., Candida (except Candida krusei), Aspergillus, and hard-to-treat molds, including Fusarium and Scedosporium In this study, we tested APX001A and comparators against 1,706 contemporary clinical fungal isolates collected in 2017 from 68 medical centers in North America (37.3%), Europe (43.4%), the Asia-Pacific region (12.7%), or Latin America (6.6%). Among the isolates tested, 78.5% were Candida spp., 3.9% were non-Candida yeasts, including 30 (1.8%) Cryptococcus neoformans var. grubii isolates, 14.7% were Aspergillus spp., and 2.9% were other molds. All isolates were tested by CLSI reference broth microdilution. APX001A (MIC50, 0.008 μg/ml; MIC90, 0.06 μg/ml) was the most active agent tested against Candida sp. isolates; corresponding anidulafungin, micafungin, and fluconazole MIC90 values were 16- to 64-fold higher. Similarly, APX001A (MIC50, 0.25 μg/ml; MIC90, 0.5 μg/ml) was ≥8-fold more active than anidulafungin, micafungin, and fluconazole against C. neoformans var. grubii Against Aspergillus spp., AXP001A (50% minimal effective concentration [MEC50], 0.015 μg/ml; MEC90, 0.03 μg/ml) was comparable in activity to anidulafungin and micafungin. Aspergillus isolates (>98%) exhibited a wild-type phenotype for the mold-active triazoles (itraconazole, posaconazole, and voriconazole). APX001A was highly active against uncommon species of Candida, non-Candida yeasts, and rare molds, including 11 isolates of Scedosporium spp. (MEC values, 0.015 to 0.06 μg/ml). APX001A demonstrated potent in vitro activity against recent fungal isolates, including echinocandin- and fluconazole-resistant strains. The extended spectrum of APX001A was also notable for its potency against many less common but antifungal-resistant strains. Further studies are in progress to evaluate the clinical utility of the methyl phosphate prodrug, APX001, in difficult-to-treat resistant fungal infections.

Urinary tract colonization is enhanced by a plasmid that regulates uropathogenic Acinetobacter baumannii chromosomal genes

Multidrug resistant (MDR) Acinetobacter baumannii poses a growing threat to global health. Research on Acinetobacter pathogenesis has primarily focused on pneumonia and bloodstream infections, even though one in five A. baumannii strains are isolated from urinary sites. In this study, we highlight the role of A. baumannii as a uropathogen. We develop the first A. baumannii catheter-associated urinary tract infection (CAUTI) murine model using UPAB1, a recent MDR urinary isolate. UPAB1 carries the plasmid pAB5, a member of the family of large conjugative plasmids that represses the type VI secretion system (T6SS) in multiple Acinetobacter strains. pAB5 confers niche specificity, as its carriage improves UPAB1 survival in a CAUTI model and decreases virulence in a pneumonia model. Comparative proteomic and transcriptomic analyses show that pAB5 regulates the expression of multiple chromosomally-encoded virulence factors besides T6SS. Our results demonstrate that plasmids can impact bacterial infections by controlling the expression of chromosomal genes.

A new reassortant clade 2.3.2.1a H5N1 highly pathogenic avian influenza virus causing recent outbreaks in ducks, geese, chickens and turkeys in Bangladesh.

A total of 15 dead or sick birds from 13 clinical outbreaks of avian influenza in ducks, geese, chickens and turkeys in 2017 in Bangladesh were examined. The presence of H5N1 influenza A virus in the affected birds was detected by RT-PCR. Phylogenetic analysis based on full-length gene sequences of all eight gene segments revealed that these recent outbreaks were caused by a new reassortant of clade 2.3.2.1a H5N1 virus, which had been detected earlier in 2015 during surveillance in live bird markets (LBMs) and wet lands. This reassortant virus acquired PB2, PB1, PA, NP and NS genes from low pathogenic avian influenza viruses mostly of non-H9N2 subtypes but retained HA, NA and M genes of the old clade 2.3.2.1a viruses. Nevertheless, the HA gene of these new viruses was 2.7% divergent from that of the old clade 2.3.2.1a viruses circulated in Bangladesh. Interestingly, similar reassortment events could be traced back in four 2.3.2.1a virus isolates of 2013 from backyard ducks. It suggests that this reassortant virus emerged in 2013, which took two years to be detected at a broader scale (i.e. in LBMs), another two years until it became widely spread in poultry and fully replaced the old viruses. Several mutations were detected in the recent Bangladeshi isolates, which are likely to influence possible phenotypic alterations such as increased mammalian adaptation, reduced susceptibility to antiviral agents and reduced host antiviral response.

Acinetobacter baumannii: Envelope Determinants That Control Drug Resistance, Virulence, and Surface Variability.

Acinetobacter baumannii has emerged as an important nosocomial pathogen, particularly for patients in intensive care units and with invasive indwelling devices. The most recent clinical isolates are resistant to several classes of clinically important antibiotics, greatly restricting the ability to effectively treat critically ill patients. The bacterial envelope is an important driver of A. baumannii disease, both at the level of battling against antibiotic therapy and at the level of protecting from host innate immune function. This review provides a comprehensive overview of key features of the envelope that interface with both the host and antimicrobial therapies. Carbohydrate structures that contribute to protecting from the host are detailed, and mutations that alter these structures, resulting in increased antimicrobial resistance, are explored. In addition, protein complexes involved in both intermicrobial and host-microbe interactions are described. Finally we discuss regulatory mechanisms that control the nature of the cell envelope and its impact on host innate immune function.

Carbon metabolism modulates the efficacy of drugs targeting the cytochrome bc1:aa3 in Mycobacterium tuberculosis

The influence of carbon metabolism on oxidative phosphorylation is poorly understood in mycobacteria. M. tuberculosis expresses two respiratory terminal oxidases, the cytochrome bc1:aa3 and the cytochrome bd oxidase, which are jointly required for oxidative phosphorylation and mycobacterial viability. The essentiality of the cytochrome bc1:aa3 for optimum growth is illustrated by its vulnerability to chemical inhibition by the clinical drug candidate Q203 and several other chemical series. The cytochrome bd oxidase is not strictly essential for growth but is required to maintain bioenergetics when the function of the cytochrome bc1:aa3 is compromised. In this study, we observed that the potency of drugs targeting the cytochrome bc1:aa3 is influenced by carbon metabolism. The efficacy of Q203 and related derivatives was alleviated by glycerol supplementation. The negative effect of glycerol supplementation on Q203 potency correlated with an upregulation of the cytochrome bd oxidase-encoding cydABDC operon. Upon deletion of cydAB, the detrimental effect of glycerol on the potency of Q203 was abrogated. The same phenomenon was also observed in recent clinical isolates, but to a lesser extent compared to the laboratory-adapted strain H37Rv. This study reinforces the importance of optimizing in vitro culture conditions for drug evaluation in mycobacteria, a factor which appeared to be particularly essential for drugs targeting the cytochrome bc1:aa3 terminal oxidase.

Molecular characterization and genotyping of recent isolate of Infectious Bronchitis Virus (IBV) in Egypt

In this study a total number of 22 organ samples (including trachea, lung and kidney) from 22 broiler farms from northern Upper Egypt were collected from Mars 2017 to June 2018 from chickens showing clear clinical and pathological signs of Infectious Bronchitis. The samples were prepared and examined by real time RT-PCR for diagnosis of IBV. A total number of 11 samples were positive (50%) which were used for further isolation on SPF eggs by three blind serial passages. Positive samples that showed the pathogenic lesions of IB (curling and dwarfing of embryos) were collected and tested with real time RT-PCR (rRT-PCR) for more confirmation then a part from S1 gene sequence was amplified by RT-PCR and the product was sequenced and the data have been compared with other related IBV strains. The results indicate that the Egyptian virus in this study has an identity percent reached up to 89% with other recent Egyptian isolates. However, it reached 67% with classical vaccine strains like H120 and variant I like CR88 strain. The lowest identity was observed with M41 strain (59%) in this study. The phylogenetic tree compared to other isolates from Middle East and worldwide showed that this isolate is related to the IBV variant 2 group closely related to IBVEg/1265B/2012 strain and the Israeli strain IS/1494/06.

Genetic and Antigenic Relatedness of Highly Pathogenic Avian Influenza H5Nx Viruses Currently Circulating in Egypt and Some H5 Commercial Vaccines

In the present study, samples were collected from wild and domestic waterfowls to detect recently circulating HPAI H5Nx viruses. Partial HA gene sequencing followed by phylogenetic analysis of the obtained sequences of five selected isolates was performed. Phylogenetic analysis revealed clustering of the three H5N8 isolates in clade 2.3.4.4b, with other Egyptian H5N8 isolates with amino acid identity percent with each other ranging from (96.9% - 99.3%). The two H5N1 isolates clustered in clade 2.2.1.2 with other recent Egyptian isolates. The amino acid identity percent between vaccinal strains was ranging from (88.2% - 89.6%) with H5N8 field isolates and (96.5% - 99%) with H5N1 field isolates. The amino acid identity percent between H5N8 and H5N1 field isolates ranging from (86.3% - 87.8%). The antigenic relatedness between the two HPAI local isolates (H5N1 and H5N8) was low (R value 24.8%) indicating major antigenic difference between the two subtypes. Also, a major subtype difference in antigenicity (R value 20.7-31.0%) was also detected between the local HPAI H5N8 isolate and the three vaccinal strains. While, a minor subtype difference (R value 38.7-68.7%) was detected among the examined H5N1 local isolate and the three vaccinal strains. Our results support that, HPAI H5N8 virus is genetically and antigenically distinct from HPAI H5N1 virus. Also, some commercial vaccines which are used to control avian influenza in Egypt are genetically and antigenically distinct from HPAI H5N8 virus currently circulating in Egypt.

CgMLST characterisation of invasive Neisseria meningitidis serogroup C and W strains associated with increasing disease incidence in the Republic of Ireland.

Introduction and aimsSince 2013 MenC and MenW disease incidence and associated mortality rates have increased in the Republic of Ireland. From 2002/2003 to 2012/2013, the average annual MenC incidence was 0.08/100,000, which increased to 0.34/100,000 during 2013/2014 to 2017/18, peaking in 2016/17 (0.72/100,000) with an associated case fatality rate (CFR) of 14.7%. MenW disease incidence has increased each year from 0.02/100,000 in 2013/2014, to 0.29/100,000 in 2017/18, with an associated CFR of 28.6%. We aimed to characterise and relate recent MenC isolates to the previously prevalent MenC:cc11 ET-15 clones, and also characterise and relate recent MenW isolates to the novel ‘Hajj’ clones.MethodsUsing WGS we characterised invasive (n = 74, 1997/98 to 2016/17) and carried (n = 16, 2016/17) MenC isolates, and invasive (n = 18, 2010/11 to 2016/17) and carried (n = 15, 2016/17) MenW isolates. Genomes were assembled using VelvethOptimiser and stored on the PubMLST Neisseria Bacterial Isolate Genome Sequence Database. Isolates were compared using the cgMLST approach.ResultsMost MenC and MenW isolates identified were cc11. A single MenC:cc11 sub-lineage contained the majority (68%, n = 19/28) of recent MenC:cc11 disease isolates and all carried MenC:cc11 isolates, which were interspersed and distinct from the historically significant ET-15 clones. MenW:cc11 study isolates clustered among international examples of both the original UK 2009 MenW:cc11, and novel 2013 MenW:cc11clones.ConclusionsWe have shown that the majority of recent MenC disease incidence was caused by strain types distinct from the MenC:cc11 ET-15 clone of the late 1990s, which still circulate but have caused only sporadic disease in recent years. We have identified that the same aggressive MenW clone now established in several other European countries, is endemic in the RoI and responsible for the recent MenW incidence increases. This data informed the National immunisation Advisory Committee, who are currently deliberating a vaccine policy change to protect teenagers.

Length of poly(A) tail affects transcript infectivity of three ZYMV symptom variants differing at only five amino acid positions

The incidence of plant virus diseases infecting important cucurbit vegetables in Korea has increased as new isolates have been introduced, associated with warming temperatures and vector movement caused by climate change. Transcript infectivity of full-length infectious clones of three new ZYMV isolates was dependent upon the length of the poly(A) tract; transcripts with 55 A residues were inefficiently infectious, whereas 60 A residues resulted in highly efficient infection and significantly reduced time to production of systemic symptoms. Sequences of isolates BR1 (MH042024), BR2 (MH042025), and BR3 (MH042026) showed 99% pair-wise identity and differed at only five amino acid positions in: HC-Pro (D134N in BR2), CI (F31 L in BR1), and 6 K2 (A24V in BR3), and two positions in NIb (T300S in BR2, H429Q in BR3). Cucurbita pepo plants inoculated with transcripts of clones with these amino acid differences showed symptoms that ranged from mild to severe. Phylogenetic analysis of these new ZYMV isolates with previously characterized isolates indicated that the new isolates had 87.8–97.5% identity to other ZYMV isolates and were most closely related to recent ZYMV isolates from Australia and Spain.

Antimicrobial Activity Evaluation of Tebipenem (SPR859), an Orally Available Carbapenem, against a Global Set of Enterobacteriaceae Isolates, Including a Challenge Set of Organisms.

The antimicrobial activity of tebipenem and other carbapenem agents were tested in vitro against a set of recent clinical isolates responsible for urinary tract infection (UTI), as well as against a challenge set. Isolates were tested by reference broth microdilution and included Escherichia coli (101 isolates), Klebsiella pneumoniae (208 isolates), and Proteus mirabilis (103 isolates) species. Within each species tested, tebipenem showed equivalent MIC50/90 values to those of meropenem (E. coli MIC50/90, ≤0.015/0.03 mg/liter; K. pneumoniae MIC50/90, 0.03/0.06 mg/liter; and P. mirabilis MIC50/90, 0.06/0.12 mg/liter) and consistently displayed MIC90 values 8-fold lower than imipenem. Tebipenem and meropenem (MIC50, 0.03 mg/liter) showed equivalent MIC50 results against wild-type, AmpC-, and/or extended-spectrum β-lactamase (ESBL)-producing isolates. Tebipenem also displayed MIC50/90 values 4- to 8-fold lower than imipenem against the challenge set. All carbapenem agents were less active (MIC50, ≥8 mg/liter) against isolates carrying carbapenemase genes. These data confirm the in vitro activity of the orally available agent tebipenem against prevalent UTI Enterobacteriaceae species, including those producing ESBLs and/or plasmid AmpC enzymes.

Three New Integration Vectors and Fluorescent Proteins for Use in the Opportunistic Human Pathogen Streptococcus pneumoniae.

Here, we describe the creation of three integration vectors, pPEPX, pPEPY and pPEPZ, for use with the opportunistic human pathogen Streptococcus pneumoniae. The constructed vectors, named PEP for Pneumococcal Engineering Platform (PEP), employ an IPTG-inducible promoter and BglBrick and BglFusion compatible multiple cloning sites allowing for fast and interchangeable cloning. PEP plasmids replicate in Escherichia coli and harbor integration sites that have homology in a large set of pneumococcal strains, including recent clinical isolates. In addition, several options of antibiotic resistance markers are available, even allowing for selection in multidrug resistant clinical isolates. The transformation efficiency of these PEP vectors as well as their ability to be expressed simultaneously was tested. Two of the three PEP vectors share homology of the integration regions with over half of the S. pneumoniae genomes examined. Transformation efficiency varied among PEP vectors based on the length of the homology regions, but all were highly transformable and can be integrated simultaneously in strain D39V. Vectors used for pneumococcal cloning are an important tool for researchers for a wide range of uses. The PEP vectors described are of particular use because they have been designed to allow for easy transfer of genes between vectors as well as integrating into transcriptionally silent areas of the chromosome. In addition, we demonstrate the successful production of several new spectrally distinct fluorescent proteins (mTurquoise2, mNeonGreen and mScarlet-I) from the PEP vectors. The PEP vectors and newly described fluorescent proteins will expand the genetic toolbox for pneumococcal researchers and aid future discoveries.