Genomic Analysis ofLegionella pneumophilain the Drinking Water System of a Large Building over 25 Years

Legionella pneumophila, the causative agent of Legionnaires' disease, is often found in the plumbing systems of buildings, from where it can be transmitted to humans via inhalation or aspiration of contaminated water drops. Annual routine water sampling from the potable water system of an occupational healthcare building in Basel over 25 years was performed in accordance with national guidelines. Overall, 309 water samples were collected at 38 time points over the period of 25 years. L. pneumophila was recovered from 120 water samples (38.8%) from 26 time points. No clinical infections were recorded during this period. Initial decontamination measures were successful for ~12 years, after which an increase in the total number of Legionella c.f.u. as well as of L. pneumophila-positive sites was noticed in 2007. Whole genome sequencing (WGS) analysis of n=123 isolates from n=113 samples showed all L. pneumophila to be sequence type 45 (sequence-based typing scheme). The isolates are closely related, with only 408 SNPs among all isolates after the bioinformatic removal of recombination events. Over the 25 years, a single lineage deriving from a recent common ancestor colonized the water system of this building. The phylogeny of isolate genomes can be interpreted as inferring good water circulation and possible recolonization from a common source after cleaning, with genome evolution and insertion/loss of large elements evident. Regular monitoring of waterlines in healthcare settings helps to identify concentrations of Legionella spp., and WGS is recommended for detailed investigation.

To investigate the prevalence of culturable and nonculturable Legionella species in hot water systems of public buildings in Japan and assess the risk factors associated with Legionella contamination in hot water systems. Legionella species were detected by conventional culture and molecular methods in 130 water samples collected from 40 buildings. A total of 26 (20.0%) water samples from 17 (42.5%) buildings were positive by culture, qualitative PCR or both methods: Legionella pneumophila and Leg. anisa were detected in four samples by a culture method, whereas 23 samples were positive by qualitative PCR, with the presence of various Legionella species confirmed by sequencing. Of these 23 samples, bacterial counts were quantifiable in 21 by real-time PCR (from 1.7 x 10(5) to 2.6 x 10(11) cells per litre). Phylogenetic analysis of amplified partial 16S rRNA gene showed close relations to various species of Legionella, including Leg. anisa and Leg. micdadei, all of which have been associated with respiratory diseases or increased antibody titres in human sera. Assessment of risk factors showed that turbidity, free chlorine concentration, iron concentration and heterotrophic plate count (HPC) were significantly associated with Legionella contamination (P < 0.05). Contamination of hot water systems of public buildings with culturable and nonculturable Legionella species may be a potential risk factor for Legionella infection in Japan. Adequate levels of chlorine, low levels of iron and HPC are important maintenance measures in the reduction of Legionella contamination in hot water systems. More than 40% of hot water systems in the Japanese public buildings examined were contaminated by not only culturable Leg. pneumophila and Leg. anisa but also by nonculturable pathogenic species. To our knowledge, this is the first report of both culturable and nonculturable Legionella contamination in hot water systems of public buildings in Japan.

Candida glabrata is a pathogen with reduced susceptibility to azoles and echinocandins. Analysis by traditional multilocus sequence typing (MLST) has recognized an increasing number of sequence types (STs), which vary with geography. Little is known about STs of C. glabrata in Australia. Here, we utilized whole genome sequencing (WGS) to study the genetic diversity of 51 Australian C. glabrata isolates and sought associations between STs over two time periods (2002–2004, 2010–2017), and with susceptibility to fluconazole by principal component analysis (PCA). Antifungal susceptibility was determined using Sensititre YeastOneTM Y010 methodology and WGS performed on the NextSeq 500 platform (Illumina) with in silico MLST STs inferred by WGS data. Single nucleotide polymorphisms (SNPs) in genes linked to echinocandin, azole and 5-fluorocytosine resistance were analyzed. Of 51 isolates, WGS identified 18 distinct STs including four novel STs (ST123, ST124, ST126, and ST127). Four STs accounted for 49% of isolates (ST3, 15.7%; ST83, 13.7%; ST7, 9.8%; ST26, 9.8%). Split-tree network analysis resolved isolates to terminal branches; many of these comprised multiple isolates from disparate geographic settings but four branches contained Australian isolates only. ST3 isolates were common in Europe, United States and now Australia, whilst ST8 and ST19, relatively frequent in the United States, were rare/absent amongst our isolates. There was no association between ST distribution (genomic similarity) and the two time periods or with fluconazole susceptibility. WGS identified mutations in the FKS1 (S629P) and FKS2 (S663P) genes in three, and one, echinocandin-resistant isolate(s), respectively. Both mutations confer phenotypic drug resistance. Twenty-five percent (13/51) of isolates were fluconazole-resistant (MIC ≥ 64 μg/ml) of which 9 (18%) had non wild-type MICs to voriconazole and posaconazole. Multiple SNPs were present in genes linked to azole resistance such as CgPDR1 and CgCDR1, as well as several in MSH2; however, SNPs occurred in both azole-susceptible and azole-resistant isolates. Although no particular SNP in these genes was definitively associated with resistance, azole-resistant/non-wild type isolates had a propensity to harbor SNPs resulting in amino acid substitutions in Pdr1 beyond the first 250 amino acid positions. The presence of SNPs may be markers of STs. Our study shows the value of WGS for high-resolution sequence typing of C. glabrata, discovery of novel STs and potential to monitor trends in genetic diversity. WGS assessment for echinocandin resistance augments phenotypic susceptibility testing.

Vital Signs: Deficiencies in Environmental Control Identified in Outbreaks of Legionnaires’ Disease—North America, 2000–2014

The diversity of Legionella pneumophila populations within single water systems is not well understood, particularly in those unassociated with cases of Legionnaires’ disease. Here, we performed genomic analysis of 235 L. pneumophila isolates obtained from 28 water samples in 13 locations within a large occupational building. Despite regular treatment, the water system of this building is thought to have been colonized by L. pneumophila for at least 30 years without evidence of association with Legionnaires’ disease cases. All isolates belonged to one of three sequence types (STs), ST27 (n=81), ST68 (n=122) and ST87 (n=32), all three of which have been recovered from Legionnaires’ disease patients previously. Pairwise single nucleotide polymorphism differences amongst isolates of the same ST were low, ranging from 0 to 19 in ST27, from 0 to 30 in ST68 and from 0 to 7 in ST87, and no homologous recombination was observed in any lineage. However, there was evidence of horizontal transfer of a plasmid, which was found in all ST87 isolates and only one ST68 isolate. A single ST was found in 10/13 sampled locations, and isolates of each ST were also more similar to those from the same location compared with those from different locations, demonstrating spatial structuring of the population within the water system. These findings provide the first insights into the diversity and genomic evolution of a L. pneumophila population within a complex water system not associated with disease.

Recently, three cases of Legionnaires’ disease were reported in three separate nursing centers in my geographic area, the state of New York. Because this was the first time I had seen this occur during my many years of working in long-term care, I felt this would be a good time to discuss the issue. The Centers for Disease Control and Prevention (CDC) has a tool kit for Legionnaires disease, from which this update has been derived. The website and other resources are included in this article. Legionnaires’ disease, caused by the microorganisms Legionella spp., was first described after an outbreak in 1976 among attendees at an American Legion convention in Philadelphia. The disease — a type of pneumonia — is very serious, killing about 1 in 10 people who contract it. Since 2000, cases of Legionnaires’ disease have increased fourfold in the United States. In 2016, the U.S. Department of Health and Human Services reported 6,100 cases in the United States. Because Legionnaires’ disease is frequently underdiagnosed, this may not represent the true incidence. In the natural environment, Legionella occurs in fresh water but becomes a health hazard when it invades water systems in buildings. Legionnaires’ disease and Pontiac fever — another disease caused by Legionella — are transmitted via small droplets of water in the air, which can be inhaled. It is not usually transmitted from person to person. The risk factors for Legionnaires’ disease include age 50 and older, a history of smoking, chronic lung disease, immune system disorders, systemic cancer, diabetes, renal failure, or hepatic failure. A recent overnight stay outside the home — including stay in a health care facility — is also a risk factor, as is exposure to hot tubs. Of more than 60 pathogenic species of Legionella, Legionella pneumophila serogroup 1 causes most cases of legionellosis (the collective term for Legionnaires’ disease and Pontiac fever). In natural aquatic systems, Legionella microorganisms grow and multiply within protozoa, single-celled microorganisms such as amoebae. Human alveolar macrophages resemble protozoa, which makes them a suitable host for Legionella in the human lungs. After an incubation period of two to 10 days (with an average of five to six days) from the time of exposure, the symptoms that manifest include:•Fever•Myalgia•Cough•Shortness of breath•Headache•Delirium•Radiograph positive for pneumonia The preferred diagnostic tests for Legionnaires’ disease are concurrent culture of lower respiratory secretions (e.g., sputum, bronchoalveolar lavage) on selective media and the Legionella urinary antigen test. Ideally, sputum should be obtained before starting an antibiotic, but the treatment should not be delayed to accommodate this. Urinary antigen testing is effective in detecting Legionella infections for days to weeks after treatment. Serological assays are not recommended due to their low specificity. •Legionella should be considered in patients who do not respond to antibiotics or are immunocompromised. Be aware of any recent outbreaks.•Macrolides and respiratory fluoroquinolones are the preferred agents for treatment (see “CDC Resources”).•The patient may require hospitalization.•Respiratory isolation is not required because transmission is via water droplets.•Supportive treatment, such as hydration and oxygen therapy, should be provided.•Resident and family education about disease transmission and treatment is advised.•All cases should be reported to the public health department.CDC Resources•Legionella (Legionnaires’ Disease and Pontiac Fever): Diagnosis, Treatment, and Prevention; https://www.cdc.gov/legionella/clinicians/diagnostic-testing.html•Legionella (Legionnaires’ Disease and Pontiac Fever): Guidelines, Standards, and Laws; https://www.cdc.gov/legionella/resources/guidelines.html•Toolkit: Developing a Water Management Program to Reduce Legionella Growth and Spread in Buildings; https://www.cdc.gov/legionella/wmp/toolkit/ •Legionella (Legionnaires’ Disease and Pontiac Fever): Diagnosis, Treatment, and Prevention; https://www.cdc.gov/legionella/clinicians/diagnostic-testing.html•Legionella (Legionnaires’ Disease and Pontiac Fever): Guidelines, Standards, and Laws; https://www.cdc.gov/legionella/resources/guidelines.html•Toolkit: Developing a Water Management Program to Reduce Legionella Growth and Spread in Buildings; https://www.cdc.gov/legionella/wmp/toolkit/ Proper maintenance of water systems in which Legionella may grow is key to disease prevention. The CDC encourages all owners of health care facilities to develop a water management plan aimed at reducing the risk of Legionella contamination. If Legionella is found in a health care facility’s water system, it must be eliminated. A tool kit developed by the CDC can help facilities determine the sources of contamination (see “CDC Resources”). Preventive measures such as cleaning the distal outlets and removing “dead legs” in the plumbing system (Long Term Living Contin Care Prof 2014:63:36–40) are known to be expensive and, unfortunately, ineffective. An outbreak of Legionella in a skilled nursing center is a disaster for everyone involved. It is critically important to recognize the signs and symptoms of a possible Legionella infection and to have a clinical policy/protocol in place for management. From an administrative prospective, follow the CDC guidelines for determining if the water system in your building is at risk for growing and spreading Legionella and learn about the newly published standards for Legionella water management and how to monitor and respond to changes in water quality. This should be included in the facility’s policy and procedure manual.

Serological and molecular identification of Legionella spp. isolated from water and surrounding air samples in Italian healthcare facilities

Infant botulism is a potentially life-threatening paralytic disease that can be associated with prolonged morbidity if not rapidly diagnosed and treated. Four infants were diagnosed and treated for infant botulism in NSW, Australia, between May 2011 and August 2013. Despite the temporal relationship between the cases, there was no close geographical clustering or other epidemiological links. Clostridium botulinum isolates, three of which produced botulism neurotoxin serotype A (BoNT/A) and one BoNT serotype B (BoNT/B), were characterized using whole-genome sequencing (WGS). In silico multilocus sequence typing (MLST) found that two of the BoNT/A-producing isolates shared an identical novel sequence type, ST84. The other two isolates were single-locus variants of this sequence type (ST85 and ST86). All BoNT/A-producing isolates contained the same chromosomally integrated BoNT/A2 neurotoxin gene cluster. The BoNT/B-producing isolate carried a single plasmid-borne bont/B gene cluster, encoding BoNT subtype B6. Single nucleotide polymorphism (SNP)-based typing results corresponded well with MLST; however, the extra resolution provided by the whole-genome SNP comparisons showed that the isolates differed from each other by >3,500 SNPs. WGS analyses indicated that the four infant botulism cases were caused by genomically distinct strains of C. botulinum that were unlikely to have originated from a common environmental source. The isolates did, however, cluster together, compared with international isolates, suggesting that C. botulinum from environmental reservoirs throughout NSW have descended from a common ancestor. Analyses showed that the high resolution of WGS provided important phylogenetic information that would not be captured by standard seven-loci MLST.

Staphylococcus aureus is an opportunistic pathogen and variable component of the human microbiota. A characteristic of atopic eczema (AE) is colonization by S. aureus, with exacerbations associated with an increased bacterial burden of the organism. Despite this, the origins and genetic diversity of S. aureus colonizing individual patients during AE disease flares is poorly understood. To examine the microevolution of S. aureus colonization, we deep sequenced S. aureus populations from nine children with moderate to severe AE and 18 non-atopic children asymptomatically carrying S. aureus nasally. Colonization by clonal S. aureus populations was observed in both AE patients and control participants, with all but one of the individuals carrying colonies belonging to a single sequence type. Phylogenetic analysis showed that disease flares were associated with the clonal expansion of the S. aureus population, occurring over a period of weeks to months. There was a significant difference in the genetic backgrounds of S. aureus colonizing AE cases versus controls (Fisher exact test, P = 0.03). Examination of intra-host genetic heterogeneity of the colonizing S. aureus populations identified evidence of within-host selection in the AE patients, with AE variants being potentially selectively advantageous for intracellular persistence and treatment resistance.

Study question Can whole transcriptome sequencing (WTS) of embryos and whole genome sequencing (WGS) of parents and embryos identify variants associated with embryo viability? Summary answer WGS/WTS can identify variants from conventional biopsies that are potentially associated with embryo viability, likely reducing failed euploid transfers. What is known already Since 45% of euploid transfers fail, and up to 95% of patients will achieve a pregnancy and live birth if enough euploid embryos are transferred, euploid failures are likely embryonic in origin. Further, they are likely due to the genetics of the embryo (beyond ploidy) since WGS studies of pregnancy loss, stillbirth and perinatal death demonstrate that 50% of euploid cases are due to single nucleotide variants (SNVs). WGS/WTS of parents and embryos has the potential to reduce failed euploid transfers by identifying SNVs that may be associated with reduced embryo viability and deprioritizing those embryos. Study design, size, duration The study was conducted on donated embryos, regardless of ploidy status. Variants were evaluated independently of ploidy, as they are expected to segregate independently. Parents who did not use donor gametes and had completed their fertility treatment were offered participation in the study. Parents were consented to the study by their treating physician and blood samples were provided for WGS. The study included 30 donated embryos from seven families (2-7 embryos/family). Participants/materials, setting, methods WGS/WTS (50x depth) was performed on at least two samples per donated embryo, including the remainder. WGS (30x depth) was performed for each parental blood sample. Genes were considered essential to embryo viability based on product of conception (PoC) and CRISPR-Cas9 knockout studies. Variants were annotated based on in vivo, in vitro, and computational evidence available in human data resources and published scientific work. Main results and the role of chance Twelve of 30 embryos (40%) were found to have predicted pathogenic, likely pathogenic or pathogenic SNVs associated with reduced embryo viability. Variants segregated independent of chromosome abnormalities, as expected. Among the 12 embryos with variants, 7 embryos had compound heterozygous SNVs in genes associated with autosomal recessive inheritance, 6 male embryos had hemizygous SNVs in genes associated with X-linked recessive inheritance (variants were confirmed to be maternally inherited) and 2 embryos had de-novo SNVs, believed to be de-novo because they were not inherited from either parent and not observed in any population databases (note the sum is greater than 12, as some embryos had multiple variants detected). Since sequencing was performed on multiple samples, the likelihood of a false positive variant call is less than 1%. Given the rate of detectable SNVs in this study is consistent with the 50% reported in the literature, we estimate that prioritizing euploid embryos without SNVs associated with reduced embryo viability could reduce failed euploid transfers by up to 50%. Further reductions could likely be achieved by including additional genetic variation, such as structural variants and copy number variants. Limitations, reasons for caution The reduction in failed euploid transfers can only be estimated at this time. Observational studies of pregnancy and live birth rates, based on transfers using WGS results are currently under way. Wider implications of the findings Reducing failed euploid transfers by prioritizing euploid embryos without SNVs that may be associated with reduced embryo viability could further improve live birth rates per embryo transfer and thereby save aspiring parents time, expense and anxiety in their IVF journeys. Trial registration number No

Whole genome sequencing of cell-free DNA for assessment of minimal residual disease in high-risk smoldering multiple myeloma

Sequence-based typing (SBT), analogous to multilocus sequence typing (MLST), is the current “gold standard” typing method for investigation of legionellosis outbreaks caused by Legionella pneumophila. However, as common sequence types (STs) cause many infections, some investigations remain unresolved. In this study, various whole-genome sequencing (WGS)-based methods were evaluated according to published guidelines, including (i) a single nucleotide polymorphism (SNP)-based method, (ii) extended MLST using different numbers of genes, (iii) determination of gene presence or absence, and (iv) a kmer-based method. L. pneumophila serogroup 1 isolates (n = 106) from the standard “typing panel,” previously used by the European Society for Clinical Microbiology Study Group on Legionella Infections (ESGLI), were tested together with another 229 isolates. Over 98% of isolates were considered typeable using the SNP- and kmer-based methods. Percentages of isolates with complete extended MLST profiles ranged from 99.1% (50 genes) to 86.8% (1,455 genes), while only 41.5% produced a full profile with the gene presence/absence scheme. Replicates demonstrated that all methods offer 100% reproducibility. Indices of discrimination range from 0.972 (ribosomal MLST) to 0.999 (SNP based), and all values were higher than that achieved with SBT (0.940). Epidemiological concordance is generally inversely related to discriminatory power. We propose that an extended MLST scheme with ∼50 genes provides optimal epidemiological concordance while substantially improving the discrimination offered by SBT and can be used as part of a hierarchical typing scheme that should maintain backwards compatibility and increase discrimination where necessary. This analysis will be useful for the ESGLI to design a scheme that has the potential to become the new gold standard typing method for L. pneumophila.

Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

Legionella pneumophila is a contaminant of man-made water systems, including potable water, cooling towers, water systems of large buildings, etc. It is the most common causative agent of legionellosis, a respiratory infection, which may give rise to restricted outbreaks. To survey environmental water samples from hospitals and private habitations in Bologna, we developed a species-specific nested and a TaqMan real-time PCR for the detection of L. pneumophila. We compared the two assays and both to cultural isolation. The targeted gene was macrophage infectivity potentiator (mip), conserved in L. pneumophila, and divergent in other legionellae. One assay was based on a nested PCR and the other on a TaqMan real-time PCR protocol. Their sensitivities were 14 % or 5% higher than that of cultural isolation respectively. The detection limits were 1-2 genome equivalents per 50-microl reaction. Specificity was assessed using DNA from nine target and 20 nontarget organisms. When applied to water samples, both assays detected L. pneumophila at 80% or higher frequency. The species-specific molecular diagnosis of L. pneumophila by means of nested PCR does not require a specific instrumentation, exhibits a high sensitivity, and is advantageous over the cultural isolation and real-time PCR detection. It allows to quickly monitor water samples for the risk assessment of environmental contaminations.

The aim of this study was to determine the prevalence of L. pneumophila in water supply systems, hospitals and public buildings in the Lublin region of eastern Poland. The study was carried out in 26 different objects in the Lublin region. The number of Legionella bacteria in water samples was determined by the membrane filtration method and/or by surface inoculation in accordance with the standards. The study showed the presence of L. pneumophila in 166 hot water samples (74.77%). In 34.33% (n=57) of water samples the count of tested bacteria exceeded the acceptable level of >100 CFU/100 ml. Of the samples where an acceptable level of bacteria was exceeded, 49 samples had an average level of L. pneumophila (100-1,000 CFU/100 ml), and the level in 8 samples was high (>1,000 CFU/100 ml). The water samples collected form the hot water supply system of hospitals and public buildings showed exceeded counts of L. pneumophila, indicating the risk of infection. The constant monitoring of water distribution systems is an important element of the control of infections caused by these organisms.