Terrisporobacter hibernicus sp. nov., isolated from bovine faeces in Northern Ireland.

A new species of Terrisporobacter, a Gram-positive, spore-forming anaerobic group, proposed name Terrisporobacter hibernicus sp. nov., was isolated in Northern Ireland from bovine faeces collected in 2016. Designated as MCA3T, cells of T. hibernicus sp. nov. are rod shaped and motile. Cells tolerate NaCl from 0.5 to 5.5 % (w/v), with a pH tolerance between pH 6 and 9. The optimal temperature for growth is 35-40 °C, and temperatures from 20 to 30 °C are tolerated. The polar lipid profile displays diphosphatidylglycerol, phosphatidylglycerol, two aminoglycolipids, one glycophospholipid, one aminolipid, three glycolipids, five phospholipids and one lipid. No respiratory quinones are detected. The predominant fatty acid profile includes C16 : 0 at 22.8 %. Strain MCA3T is positive for glucose and maltose acidification, as well as glycerol and sorbitol. The biochemical results from a VITEK2 assay of strain MCA3T, Terrisporobacter petrolearius LAM0A37T and Terrisporobacter mayombei DSM 6539T are also included for the first time. The closed and complete genome of strain MCA3T from a hybrid Oxford Nanopore Technology MinION/Illumina assembly reveals no evidence for known virulence genes. Draft genome sequencing of T. mayombei DSM 6539T and T. petrolearius LAM0A37T, as performed by Illumina MiSeq, provides reference genomes for these respective species of Terrisporobacter for the first time. DNA-DNA hybridization values (d4) of MCA3T to Terrisporobacter glycolicus ATCC 14880T, T. petrolearius LAM0A37T and T. mayombei DSM 6539T are 48.8, 67.4 and 46.3 %, with cutoff value at 70 %. The type strain for T. hibernicus sp. nov. is MCA3T (=NCTC 14625T=LMG 32430T).

The putative bacterial oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) suppresses antibiotic resistance and pathogenicity in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an extracellular opportunistic bacterial pathogen commonly associated with infectious complications in susceptible individuals, such as those with underlying diseases including HIV/AIDS and cystic fibrosis. Antibiotic resistance in multiple strains of P. aeruginosa is a rapidly developing clinical problem. We have previously demonstrated that the oxygen levels at the site of P. aeruginosa infection can strongly influence virulence and antibiotic resistance in this pathogen, although the oxygen-sensing and -signaling mechanisms underpinning this response have remained unknown. In this study, we investigated the potential role of the putative oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) in the control of virulence and antibiotic resistance in P. aeruginosa We found that a P. aeruginosa strain lacking PPHD (PAO310) exhibits increased virulence associated with increased bacterial motility. Furthermore, PPHD-deficient P. aeruginosa displayed enhanced antibiotic resistance against tetracycline through increased expression of the xenobiotic transporters mexEF-oprN and MexXY. Of note, the effect of the PPHD knockout on antibiotic resistance was phenocopied in bacteria exposed to atmospheric hypoxia. We conclude that PPHD is a putative bacterial oxygen sensor that may link microenvironmental oxygen levels to virulence and antibiotic resistance in P. aeruginosa.

Whole-genome Sequencing to Track Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Transmission in Nosocomial Outbreaks.

BACKGROUND: During the first wave of the coronavirus disease 2019 (COVID-19) pandemic, outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in healthcare institutions posed a significant problem. Due to limited evidence, guidance on appropriate infection prevention and control (IPC) measures such as the wearing of face masks varied. Here, we applied whole virus genome sequencing (WvGS) to analyze transmission routes of SARS-CoV-2 in hospital-acquired (HA) COVID-19. METHODS: An investigation was undertaken for all HA cases of COVID-19 from March to April 2020. Fifty SARS-CoV-2 samples were analysed by WvGS and their phylogenetic relationship established. RESULTS: WvGS identified transmission events previously undetected by epidemiological analysis and provided evidence for SARS-CoV-2 transmission between healthcare workers (HCW) and patients and among HCW themselves. The majority of HA COVID-19 cases occurred in patients highly dependent on nursing care, suggesting the likely route of transmission was by close contact or droplet, rather than aerosol, transmission. Mortality among HA COVID-19 infections was recorded as 33%. CONCLUSIONS: This study provides evidence that SARS-CoV-2 transmission occurs from symptomatic and asymptomatic HCWs to patients. Interventions including comprehensive screening of HCWs for COVID-19 symptoms, PCR testing of asymptomatic HCWs upon identification of HA cases and implementation of universal use of surgical masks for all clinical care is indicated to prevent viral transmission. Our study highlights the importance of close collaboration between guidance bodies and frontline IPC experts for developing control measures in an emergency pandemic situation caused by a virus with undefined transmission modus.

Genomic Analysis of Mycobacterium abscessus Complex Isolates Collected in Ireland between 2006 and 2017.

Members of the Mycobacterium abscessus complex (MABC) are multidrug-resistant nontuberculous mycobacteria and cause opportunistic pulmonary infections in individuals with cystic fibrosis (CF). In this study, genomic analysis of MABC isolates was performed to gain greater insights into the epidemiology of circulating strains in Ireland. Whole-genome sequencing (WGS) was performed on 70 MABC isolates that had been referred to the Irish Mycobacteria Reference Laboratory between 2006 and 2017 across nine Irish health care centers. The MABC isolates studied comprised 52 isolates from 27 CF patients and 18 isolates from 10 non-CF patients. WGS identified 57 (81.4%) as M. abscessus subsp. abscessus, 10 (14.3%) as M. abscessus subsp. massiliense, and 3 (4.3%) as M. abscessus subsp. bolletii Forty-nine (94%) isolates from 25 CF patients were identified as M. abscessus subsp. abscessus, whereas 3 (6%) isolates from 2 CF patients were identified as M. abscessus subsp. massiliense Among the isolates from non-CF patients, 44% (8/18) were identified as M. abscessus subsp. abscessus, 39% (7/18) were identified as M. abscessus subsp. massiliense, and 17% (3/18) were identified as M. abscessus subsp. bolletii WGS detected two clusters of closely related M. abscessus subsp. abscessus isolates that included isolates from different CF centers. There was a greater genomic diversity of MABC isolates among the isolates from non-CF patients than among the isolates from CF patients. Although WGS failed to show direct evidence of patient-to-patient transmission among CF patients, there was a predominance of two different strains of M. abscessus subsp. abscessus Furthermore, some MABC isolates were closely related to global strains, suggesting their international spread. Future prospective real-time epidemiological and clinical data along with contemporary MABC sequence analysis may elucidate the sources and routes of transmission among patients infected with MABC.

Yersinia canariae sp. nov., isolated from a human yersiniosis case.

A Gram-negative rod from the Yersinia genus was isolated from a clinical case of yersiniosis in the United Kingdom. Long read sequencing data from an Oxford Nanopore Technologies (ONT) MinION in conjunction with Illumina HiSeq reads were used to generate a finished quality genome of this strain. Overall Genome Related Index (OGRI) of the strain was used to determine that it was a novel species within Yersinia, despite biochemical similarities to Yersinia enterocolitica. The 16S ribosomal RNA gene accessions are MN434982-MN434987 and the accession number for the complete and closed chromosome is CP043727. The type strain is SRR7544370T (=NCTC 14382T/=LMG 31573T).

A Core Genome Multilocus Sequence Typing Scheme for Enterococcus faecalis.

Among enterococci, Enterococcus faecalis occurs ubiquitously, with the highest incidence of human and animal infections. The high genetic plasticity of E. faecalis complicates both molecular investigations and phylogenetic analyses. Whole-genome sequencing (WGS) enables unraveling of epidemiological linkages and putative transmission events between humans, animals, and food. Core genome multilocus sequence typing (cgMLST) aims to combine the discriminatory power of classical multilocus sequence typing (MLST) with the extensive genetic data obtained by WGS. By sequencing a representative collection of 146 E. faecalis strains isolated from hospital outbreaks, food, animals, and colonization of healthy human individuals, we established a novel cgMLST scheme with 1,972 gene targets within the Ridom SeqSphere+ software. To test the E. faecalis cgMLST scheme and assess the typing performance, different collections comprising environmental and bacteremia isolates, as well as all publicly available genome sequences from the NCBI and SRA databases, were analyzed. In more than 98.6% of the tested genomes, >95% good cgMLST target genes were detected (mean, 99.2% target genes). Our genotyping results not only corroborate the known epidemiological background of the isolates but exceed previous typing resolution. In conclusion, we have created a powerful typing scheme, hence providing an international standardized nomenclature that is suitable for surveillance approaches in various sectors, linking public health, veterinary public health, and food safety in a true One Health fashion.

Yersinia hibernica sp. nov., isolated from pig-production environments.

A Gram-stain-negative, rod-shaped strain isolated from pig-production environments was identified as a new species within the genus Yersinia using multifaceted genomic and biochemical approaches. The genome of this strain was closed using a hybrid assembly approach combining both high accuracy short read sequencing data with long read sequencing technology. Phylogenetic analysis of the 16S rRNA gene showed ~98 % similarity to Yersinia kristensenii and ~98 % similarity to Yersinia enterocolitica. Average nucleotide identity (OrthoANI) values were calculated as 85.79 % to Y. kristensenii ATCC 33638T and 85.73 % to Y. enterocolitica ATCC 9610T thereby providing evidence that this isolate should be considered as a novel species. The type strain is CFS1934T (=NCTC 14222T=LMG 31076T).

Update on prevalence and mechanisms of resistance to linezolid, tigecycline and daptomycin in enterococci in Europe: Towards a common nomenclature.

Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens. Invasive VRE infections are difficult to treat since common therapeutic options including ampicillin and glycopeptides often fail. In vitro, most VRE remain susceptible to last-resort antibiotics such as linezolid, tigecycline and daptomycin. However, neither tigecycline nor linezolid act in a bactericidal manner, and daptomycin has proven activity only at high dosages licensed for treating enterococcal endocarditis. Despite these pharmacological and therapeutic limitations, reports on resistance to these last-resort drugs in VRE, and enterococci in general, have increased in recent years. In this review, we briefly recapitulate the current knowledge on the mode of action as well as the known and novel mechanisms of resistance and describe surveillance data on resistance to linezolid, tigecycline and daptomycin in enterococci. In addition, we also suggest a common nomenclature for designating enterococci and VRE with resistances to these important last-resort antibiotics.

Macrophage migration inhibitory factor enhances Pseudomonas aeruginosa biofilm formation, potentially contributing to cystic fibrosis pathogenesis.

Macrophage migration inhibitory factor (MIF) is a key proinflammatory mediator that we have previously shown to be associated with an aggressive clinical phenotype in cystic fibrosis. It possesses unique tautomerase enzymatic activity. However, to date, no human-derived substrate has been identified that has the capacity to interact with this cytokine's unique tautomerase activity. This led us to hypothesize that MIF may have the capacity to interact with external substrates. We describe for the first time how Pseudomonas aeruginosa can utilize human recombinant MIF (rMIF) to significantly (P < 0.01) enhance its endogenous biofilm formation. Our in vivo studies demonstrate that utilizing a small-molecular-weight inhibitor targeting MIF's tautomerase activity (SCD-19) significantly reduces the inflammatory response in a murine pulmonary chronic P. aeruginosa model. In addition, we show that in in vitro experiments, pretreatment of P. aeruginosa with rMIF is associated with reduced bacterial killing by tobramycin. Our novel findings support the concept of an anti-MIF strategy that targets this enzymatic activity as a potential future antibacterial therapeutic approach.-Tynan, A., Mawhinney, L., Armstrong, M. E., O'Reilly, C., Kennedy, S., Caraher, E., Jülicher, K., O'Dwyer, D., Maher, L., Schaffer, K., Fabre, A., McKone, E. F., Leng, L., Bucala, R., Bernhagen, J., Cooke, G., Donnelly, S. C. Macrophage migration inhibitory factor enhances Pseudomonas aeruginosa biofilm formation, potentially contributing to cystic fibrosis pathogenesis.

Hypoxia Reduces the Pathogenicity of Pseudomonas aeruginosa by Decreasing the Expression of Multiple Virulence Factors.

Our understanding of how the course of opportunistic bacterial infection is influenced by the microenvironment is limited. We demonstrate that the pathogenicity of Pseudomonas aeruginosa strains derived from acute clinical infections is higher than that of strains derived from chronic infections, where tissues are hypoxic. Exposure to hypoxia attenuated the pathogenicity of strains from acute (but not chronic) infections, implicating a role for hypoxia in regulating bacterial virulence. Mass spectrometric analysis of the secretome of P. aeruginosa derived from an acute infection revealed hypoxia-induced repression of multiple virulence factors independent of altered bacterial growth. Pseudomonas aeruginosa lacking the Pseudomonas prolyl-hydroxylase domain-containing protein, which has been implicated in bacterial oxygen sensing, displays reduced virulence factor expression. Furthermore, pharmacological hydroxylase inhibition reduces virulence factor expression and pathogenicity in a murine model of pneumonia. We hypothesize that hypoxia reduces P. aeruginosa virulence at least in part through the regulation of bacterial hydroxylases.

A universal stress protein upregulated by hypoxia has a role in Burkholderia cenocepacia intramacrophage survival: Implications for chronic infection in cystic fibrosis.

Universal stress proteins (USPs) are ubiquitously expressed in bacteria, archaea, and eukaryotes and play a lead role in adaptation to environmental conditions. They enable adaptation of bacterial pathogens to the conditions encountered in the human niche, including hypoxia, oxidative stress, osmotic stress, nutrient deficiency, or acid stress, thereby facilitating colonization. We previously reported that all six USP proteins encoded within a low-oxygen activated (lxa) locus in Burkholderia cenocepacia showed increased abundance during chronic colonization of the cystic fibrosis (CF) lung. However, the role of USPs in chronic cystic fibrosis infection is not well understood. Structural modeling identified surface arginines on one lxa-encoded USP, USP76, which suggested it mediated interactions with heparan sulfate. Using mutants derived from the B. cenocepacia strain, K56-2, we show that USP76 is involved in host cell attachment. Pretreatment of lung epithelial cells with heparanase reduced the binding of the wild-type and complement strains but not the Δusp76 mutant strain, indicating that USP76 is directly or indirectly involved in receptor recognition on the surface of epithelial cells. We also show that USP76 is required for growth and survival in many conditions associated with the CF lung, including acidic conditions and oxidative stress. Moreover, USP76 also has a role in survival in macrophages isolated from people with CF. Overall, while further elucidation of the exact mechanism(s) is required, we can conclude that USP76, which is upregulated during chronic infection, is involved in bacterial survival within CF macrophages, a hallmark of Burkholderia infection.

Hypoxia increases antibiotic resistance in Pseudomonas aeruginosa through altering the composition of multidrug efflux pumps.

Antibiotic resistance is a significant and developing problem in general medical practice and a common clinical complication in cystic fibrosis patients infected with Pseudomonas aeruginosa. Such infections occur within hypoxic mucous deposits in the cystic fibrosis lung; however, little is known about how the hypoxic microenvironment influences pathogen behavior. Here we investigated the impact of hypoxia on antibiotic resistance in P. aeruginosa. The MICs of a selection of antibiotics were determined for P. aeruginosa grown under either normoxic or hypoxic conditions. The expression of mRNAs for resistance-nodulation-cell division (RND) multidrug efflux pump linker proteins was determined by real-time PCR, and multidrug efflux pump activity was inhibited using Phe-Arg ß-naphthylamide dihydrochloride. The MIC values of a subset of clinically important P. aeruginosa antibiotics were higher for bacteria incubated under hypoxia than under normoxia. Furthermore, hypoxia altered the stoichiometry of multidrug efflux pump linker protein subtype expression, and pharmacologic inhibition of these pumps reversed hypoxia-induced antibiotic resistance. We hypothesize that hypoxia increases multidrug resistance in P. aeruginosa by shifting multidrug efflux pump linker protein expression toward a dominance of MexEF-OprN. Thus, microenvironmental hypoxia may contribute significantly to the development of antibiotic resistance in P. aeruginosa infecting cystic fibrosis patients.

Surveillance of Epstein-Barr virus loads in adult liver transplantation: associations with age, sex, posttransplant times, and transplant indications.

Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disorder (PTLD) is a life-threatening complication after adult orthotopic liver transplantation (AOLT). Besides EBV and immunosuppression, relatively little is known about the pretransplant clinical parameters associated with the risk of PTLD, and the benefit of using EBV surveillance to predict EBV-associated disease in AOLT patients is uncertain. The aims of this single-center study were to monitor EBV viral loads (VLs) in AOLT patients and to investigate any associations with age, sex, cytomegalovirus (CMV) serostatus, posttransplant times, and indications for transplantation. 1275 blood samples that were collected from 197 AOLT patients 1 day to more than 15 years after transplantation were investigated with quantitative polymerase chain reaction for EBV and CMV DNA. Seventy-two percent of the patients had EBV DNAemia less than 100 days after transplantation without clinical manifestations. No association was observed between the EBV copy numbers and the time since transplantation. EBV DNAemia was weakly associated with male sex but was not associated with age, CMV serostatus, or indications for AOLT. The highest EBV VL levels were observed in patients who presented with congenital liver diseases, whereas patients with viral hepatitis maintained high EBV VLs after transplantation. None of the patients developed PTLD during the study period; however, 3 patients presented with EBV-associated diseases. In conclusion, EBV DNAemia is common in AOLT patients, and routine EBV surveillance has limited value for predicting EBV-associated morbidity or mortality.

Draft Genome Sequence and Annotation of Acinetobacter soli AS15, Isolated from an Irish Hospital.

We report the draft genome sequence of Acinetobacter soli AS15, which was isolated in 2018 from a rectal screen of a patient at St. Vincent's University Hospital (Dublin, Ireland). The draft genome sequence is 3,589,002 bp and was assembled into 82 contigs.

Kinetics of host immune responses and cytomegalovirus resistance in a liver transplant patient.

Among solid organ transplant (SOT) recipients, donor-seropositive/recipient-seronegative (D+/R-) cytomegalovirus (CMV) status is associated with the highest risk of ganciclovir-resistant CMV disease, which has been reported for patients receiving oral ganciclovir but not valganciclovir prophylaxis. We report a case of CMV breakthrough infection in a D+/R- liver transplant patient while he was receiving oral valganciclovir. Forty samples collected over 6 months were analyzed for the CMV viral load, lymphocyte counts, cytokine levels, and lymphocyte differentiation status. Genotypic resistance testing of the viral UL97 gene was performed when the patient failed to respond. CMV viremia occurred on day 50 post-transplant, and 5 samples taken between days 50 and 85 showed the wild-type UL97 genotype. The appearance of deletion 594-595 was observed from day 114 post-transplant. Viral loads declined when foscarnet was commenced and remained below 10,000 copies/mL when the lymphocyte count was greater than 1000/microL (P = 0.02). T cell responses revealed significant expansion of CD8+ terminal effector memory cells. CD4+ cells were largely populations of naïve and central memory cells. Circulating interleukin 10 (IL-10) levels correlated with the viral load (P < 0.0001). Seroconversion occurred on day 230. The CMV viral load in combination with lymphocyte counts and IL-10 may be a predictive marker for the risk of development of resistant CMV disease in D+/R- SOT patients.

An Unusual Case of Brucella abortus Prosthetic Joint Infection.

Brucellosis is a systemic infection caused by brucella species. Prosthetic joint infection due to brucella species is rare. We report the case of a prosthetic joint infection presenting fourteen years post treatment for systemic brucellosis.

Increased Virulence of Bloodstream Over Peripheral Isolates of P. aeruginosa Identified Through Post-transcriptional Regulation of Virulence Factors.

The factors influencing the virulence of P. aeruginosa in the development of invasive infection remain poorly understood. Here, we investigated the role of the host microenvironment in shaping pathogen virulence and investigated the mechanisms involved. Comparing seven paired genetically indistinguishable clinical bloodstream and peripheral isolates of P. aeruginosa, we demonstrate that isolates derived from bloodstream infections are more virulent than their peripheral counterparts (p = 0.025). Bloodstream and peripheral isolates elicited similar NF-kB responses in a THP-1 monocyte NF-kappaB reporter cell line implicating similar immunogenicity. Proteomic analysis by mass spectrometry identified multiple virulence and virulence-related factors including LecA and RpoN in significantly greater abundance in the bacterial supernatant from the bloodstream isolate in comparison to that from the corresponding peripheral isolate. Investigation by qPCR revealed that control of expression of these virulence factors was not due to altered levels of transcription. Based on these data, we hypothesize a post-transcriptional mechanism of virulence regulation in P. aeruginosa bloodstream infections influenced by surrounding microenvironmental conditions.

The involvement of the low-oxygen-activated locus of Burkholderia cenocepacia in adaptation during cystic fibrosis infection.

Chronic infection with opportunistic pathogens including Burkholderia cepacia complex (Bcc) is a hallmark of cystic fibrosis (CF). We investigated the adaptive mechanisms facilitating chronic lung infection in sequential Bcc isolates from two siblings with CF (P1 and P2), one of whom also experienced intermittent blood-stream infections (P2). We previously showed increased lung cell attachment with colonisation time in both P1 and P2. WGS analysis confirmed that the isolates are closely related. Twelve genes showed three or more mutations, suggesting these were genes under selection. Single nucleotide polymorphisms (SNVs) in 45 regulatory genes were also observed. Proteomic analysis showed that the abundance of 149 proteins increased over 61-months in sputum isolates, and both time- and source-related alterations in protein abundance between the second patient's isolates. A consistent time-dependent increase in abundance of 19 proteins encoded by a low-oxygen-activated (lxa) locus was observed in both sets of isolates. Attachment was dramatically reduced in a B. cenocepacia K56-2Δlxa-locus deletion mutant, further indicating that it encodes protein(s) involved in host-cell attachment. Time-related changes in virulence in Galleria mellonella or motility were not observed. We conclude that the lxa-locus, associated with anoxic persistence in vitro, plays a role in host-cell attachment and adaptation to chronic colonization in the hypoxic niche of the CF lung.

The impact of hypoxia on bacterial infection.

Tissue hypoxia is a common microenvironmental feature during inflammation associated with bacterial infection. Hypoxia has recently been shown to play an important role in both innate and adaptive host immunity through the regulation of transcription factors, including hypoxia-inducible factor and nuclear factor-κB, in both infiltrating immunocytes and inflamed resident cells. Recent studies have suggested that, by regulating these important immune effector pathways in host tissues, hypoxia can significantly alter the process of bacterial infection and subsequent disease progression. Although hypoxia is often beneficial in terms of reducing the development of infection, its net effect depends on a number of factors, including the nature of the pathogen and the characteristics of the infection encountered. In this minireview, we will discuss the impact of local tissue hypoxia and the resulting activation of hypoxia-sensitive pathways on bacterial infection by a range of pathogens. Furthermore, we will review how this knowledge may be used to develop new approaches to anti-infective therapeutics.