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The 16S rRNA gene has been a mainstay of sequence-based bacterial analysis for decades. However, high-throughput sequencing of the full gene has only recently become a realistic prospect. Here, we use in silico and sequence-based experiments to critically re-evaluate the potential of the 16S gene to provide taxonomic resolution at species and strain level. We demonstrate that targeting of 16S variable regions with short-read sequencing platforms cannot achieve the taxonomic resolution afforded by sequencing the entire (~1500 bp) gene. We further demonstrate that full-length sequencing platforms are sufficiently accurate to resolve subtle nucleotide substitutions (but not insertions/deletions) that exist between intragenomic copies of the 16S gene. In consequence, we argue that modern analysis approaches must necessarily account for intragenomic variation between 16S gene copies. In particular, we demonstrate that appropriate treatment of full-length 16S intragenomic copy variants has the potential to provide taxonomic resolution of bacterial communities at species and strain level. Here, the authors explore the potential of the 16S gene for discriminating bacterial taxa and show that full-length sequencing combined with appropriate clustering of intragenomic sequence variation can provide accurate representation of bacterial species in microbiome datasets.

The recent development of a system for long-term in vitro culture of the syphilis spirochete, Treponema pallidum subsp. pallidum , has introduced the possibility of detailed genetic analysis of this bacterium. In this study, the in vitro culture system was used to isolate and characterize clonal populations of T . pallidum subsp. pallidum Nichols, the most widely studied strain. In limiting dilutions experiments, it was possible to establish cultures with inocula as low as 0.5 T . pallidum per well despite the long generation time (~35 to 40 hours) of this organism. Six Nichols strain clones isolated by limiting dilution were characterized in detail. All clones exhibited indistinguishable morphology and motility, highly similar in vitro multiplication rates, and comparable infectivity in the rabbit model (ID50 ≤ 100 bacteria). Genomic sequencing revealed sequence heterogeneity in the form of insertions or deletions at 5 sites, single nucleotide variations at 20 sites, and polynucleotide (polyG/C) tract length differences at 22 locations. Genomic sequences of the uncloned Nichols strain preparations propagated in rabbits or in vitro cultures exhibited substantial heterogeneity at these locations, indicating coexistence of many varied ‘clonotypes’ within these populations. Nearly all genetic variations were specific for the Nichols strain and were not detected in the >280 T . pallidum genomic sequences that are currently available. We hypothesize that these Nichols strain-specific sequence variations arose independently either during human infection or within the 110 years since the strain’s initial isolation, and thus represent examples of microevolution and divergence.

In the last decade livestock-associated methicillin-resistant S. aureus (LA-MRSA) has become a public health concern in many parts of the world. Sequence type 398 (ST398) has been the most commonly reported type of LA-MRSA. While many studies have focused on long-term exposure experienced by swine workers, this study focuses on short-term exposures experienced by veterinary students conducting diagnostic investigations. The objectives were to assess the rate of MRSA acquisition and longevity of carriage in students exposed to pork farms and characterize the recovered MRSA isolates. Student nasal swabs were collected immediately before and after farm visits. Pig nasal swabs and environmental sponge samples were also collected. MRSA isolates were identified biochemically and molecularly including spa typing and antimicrobial susceptibility testing. Thirty (30) veterinary students were enrolled and 40 pork farms were visited. MRSA was detected in 30% of the pork farms and in 22% of the students following an exposure to a MRSA-positive pork farm. All students found to be MRSA-positive initially following farm visit were negative for MRSA within 24 hours post visit. Most common spa types recovered were t002 (79%), t034 (16%) and t548 (4%). Spa types found in pork farms closely matched those recovered from students with few exceptions. Resistance levels to antimicrobials varied, but resistance was most commonly seen for spectinomycin, tetracyclines and neomycin. Non-ST398 MRSA isolates were more likely to be resistant to florfenicol and neomycin as well as more likely to be multidrug resistant compared to ST398 MRSA isolates. These findings indicate that MRSA can be recovered from persons visiting contaminated farms. However, the duration of carriage was very brief and most likely represents contamination of nasal passages rather than biological colonization. The most common spa types found in this study were associated with ST5 and expands the range of livestock-associated MRSA types.

Background Changes in diet and exercise can alter the gut microbiome of humans and mice; however, few studies to date have assessed the microbiomes of highly fit athletes. In this pilot study, we used metagenomic whole genome shotgun (mWGS) and metatranscriptomic (RNA-Seq) sequencing to show what organisms are both present and active in the gut microbiomes of both professional and amateur level competitive cyclists and to determine if any significant differences exist between these two groups. Results Using mWGS sequencing data, we showed that the gut microbiomes of 33 cyclists split into three taxonomic clusters, characterized by either high Prevotella , high Bacteroides or a mix of many genera including Bacteroides , Prevotella , Eubacterium , Ruminococcus , and Akkermansia . While no significant correlations could be found between taxonomic cluster and being either a professional or amateur level cyclist, high abundance of the genus Prevotella (≥2.5%) was significantly correlated with time reported exercising during an average week. Increased abundance of Prevotella was correlated with a number of amino acid and carbohydrate metabolism pathways, including branched chain amino acid metabolism. Further analysis of the metatranscriptome revealed significant taxonomic differences when compared to the metagenome. There was increased abundance of Methanobrevibacter smithii transcripts in a number of professional cyclists in comparison to amateur cyclists and this archaeon had upregulation of genes involved in the production of methane. Furthermore, when methane metabolism was upregulated, there was similar upregulation of energy and carbohydrate metabolism pathways. Conclusions These results provide a framework for common constituents of the gut community in individuals who follow an exercise-rich lifestyle. These data also suggest how certain organisms such as M. smithii may beneficially influence the metabolic efficiency of the gut community in professional cyclists due to synergistic metabolic cross-feeding events.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a global threat. We therefore analysed the bacterial characteristics of CRKP infections and the clinical outcomes of patients with CRKP infections across different countries. In this prospective, multicentre, cohort study (CRACKLE-2), hospitalised patients with cultures positive for CRKP were recruited from 71 hospitals in Argentina, Australia, Chile, China, Colombia, Lebanon, Singapore, and the USA. The first culture positive for CRKP was included for each unique patient. Clinical data on post-hospitalisation death and readmission were collected from health records, and whole genome sequencing was done on all isolates. The primary outcome was a desirability of outcome ranking at 30 days after the index culture, and, along with bacterial characteristics and 30-day all-cause mortality (a key secondary outcome), was compared between patients from China, South America, and the USA. The desirability of outcome ranking was adjusted for location before admission, Charlson comorbidity index, age at culture, Pitt bacteremia score, and anatomical culture source through inverse probability weighting; mortality was adjusted for the same confounders, plus region where relevant, through multivariable logistic regression. This study is registered at ClinicalTrials.gov, NCT03646227, and is complete. Between June 13, 2017, and Nov 30, 2018, 991 patients were enrolled, of whom 502 (51%) met the criteria for CRKP infection and 489 (49%) had positive cultures that were considered colonisation. We observed little intra-country genetic variation in CRKP. Infected patients from the USA were more acutely ill than were patients from China or South America (median Pitt bacteremia score 3 [IQR 2–6] vs 2 [0–4] vs 2 [0–4]) and had more comorbidities (median Charlson comorbidity index 3 [IQR 2–5] vs 1 [0–3] vs 1 [0–2]). Adjusted desirability of outcome ranking outcomes were similar in infected patients from China (n=246), South America (n=109), and the USA (n=130); the estimates were 53% (95% CI 42–65) for China versus South America, 50% (41–61) for the USA versus China, and 53% (41–66) for the USA versus South America. In patients with CRKP infections, unadjusted 30-day mortality was lower in China (12%, 95% CI 8–16; 29 of 246) than in the USA (23%, 16–30; 30 of 130) and South America (28%, 20–37; 31 of 109). Adjusted 30-day all-cause mortality was higher in South America than in China (adjusted odds ratio [aOR] 4·82, 95% CI 2·22–10·50) and the USA (aOR 3·34, 1·50–7·47), with the mortality difference between the USA and China no longer being significant (aOR 1·44, 0·70–2·96). Global CRKP epidemics have important regional differences in patients’ baseline characteristics and clinical outcomes, and in bacterial characteristics. Research findings from one region might not be generalisable to other regions. The National Institutes of Health.

In order to examine the prevalence of Staphylococcus aureus on retail pork, three hundred ninety-five pork samples were collected from a total of 36 stores in Iowa, Minnesota, and New Jersey. S. aureus was isolated from 256 samples (64.8%, 95% confidence interval [CI] 59.9%-69.5%). S. aureus was isolated from 67.3% (202/300) of conventional pork samples and from 56.8% (54/95) of alternative pork samples (labeled \"raised without antibiotics\" or \"raised without antibiotic growth promotants\"). Two hundred and thirty samples (58.2%, 95% CI 53.2%-63.1%) were found to carry methicillin-sensitive S. aureus (MSSA). MSSA was isolated from 61.0% (183/300) of conventional samples and from 49.5% (47/95) of alternative samples. Twenty-six pork samples (6.6%, 95% CI 4.3%-9.5%) carried methicillin-resistant S. aureus (MRSA). No statistically significant differences were observed for the prevalence of S. aureus in general, or MSSA or MRSA specifically, when comparing pork products from conventionally raised swine and swine raised without antibiotics, a finding that contrasts with a prior study from The Netherlands examining both conventional and \"biologic\" meat products. In our study spa types associated with \"livestock-associated\" ST398 (t034, t011) were found in 26.9% of the MRSA isolates, while 46.2% were spa types t002 and t008--common human types of MRSA that also have been found in live swine. The study represents the largest sampling of raw meat products for MRSA contamination to date in the U.S. MRSA prevalence on pork products was higher than in previous U.S.-conducted studies, although similar to that in Canadian studies.

Though rare, breast implant-associated anaplastic large cell lymphoma (BIA-ALCL), a CD30+ T-cell lymphoma associated with textured breast implants, has adversely impacted our perception of the safety of breast implants. Its etiology unknown, one hypothesis suggests an initiating inflammatory stimulus, possibly infectious, triggers BIA-ALCL. We analyzed microbiota of breast, skin, implant and capsule in BIA-ALCL patients (n = 7), and controls via culturing methods, 16S rRNA microbiome sequencing, and immunohistochemistry. Alpha and beta diversity metrics and relative abundance of Gram-negative bacteria were calculated, and phylogenetic trees constructed. Staphylococcus spp ., the most commonly cultured microbes, were identified in both the BIA-ALCL and contralateral control breast. The diversity of bacterial microbiota did not differ significantly between BIA-ALCL and controls for any material analyzed. Further, there were no significant differences in the relative abundance of Gram-negative bacteria between BIA-ALCL and control specimens. Heat maps suggested substantial diversity in the composition of the bacterial microbiota of the skin, breast, implant and capsule between patients with no clear trend to distinguish BIA-ALCL from controls. While we identified no consistent differences between patients with BIA-ALCL-affected and contralateral control breasts, this study provides insights into the composition of the breast microbiota in this population.

Abstract Background Carbapenem resistance is a critical healthcare challenge worldwide. Particularly concerning is the widespread dissemination of Klebsiella pneumoniae carbapenemase (KPC). Klebsiella pneumoniae harboring blaKPC (KPC-Kpn) is endemic in many areas including the United States, where the epidemic was primarily mediated by the clonal dissemination of Kpn ST258. We postulated that the spread of blaKPC in other regions occurs by different and more complex mechanisms. To test this, we investigated the evolution and dynamics of spread of KPC-Kpn in Colombia, where KPC became rapidly endemic after emerging in 2005. Methods We sequenced the genomes of 133 clinical isolates recovered from 24 tertiary care hospitals located in 10 cities throughout Colombia, between 2002 (before the emergence of KPC-Kpn) and 2014. Phylogenetic reconstructions and evolutionary mapping were performed to determine temporal and genetic associations between the isolates. Results Our results indicate that the start of the epidemic was driven by horizontal dissemination of mobile genetic elements carrying blaKPC-2, followed by the introduction and subsequent spread of clonal group 258 (CG258) isolates containing blaKPC-3. Conclusions The combination of 2 evolutionary mechanisms of KPC-Kpn within a challenged health system of a developing country created the “perfect storm” for sustained endemicity of these multidrug-resistant organisms in Colombia.

Influenza A(H5N1) in Wastewater in 10 U.S. CitiesAvian influenza A(H5N1) virus is spreading through dairy herds across the United States. In this report, wastewater surveillance shows the increase of H5N1 across 10 urban communities.

The prevalence of carbapenem-resistant Klebsiella pneumoniae (CR Kp ) infections in nosocomial settings remains a public health challenge. High-risk clones such as clonal group 258 (CG258) are particularly concerning due to their association with bla KPC carriage, which can severely complicate antimicrobial treatments. Carbapenem-resistant Klebsiella pneumoniae (CR Kp ) is an urgent public health threat. Worldwide dissemination of CR Kp has been largely attributed to clonal group (CG) 258. However, recent evidence indicates the global emergence of a CR Kp CG307 lineage. Houston, TX, is the first large city in the United States with detected cocirculation of both CR Kp CG307 and CG258. We sought to characterize the genomic and clinical factors contributing to the parallel endemic spread of CG258 and CG307. CR Kp isolates were collected as part of the prospective, Consortium on Resistance against Carbapenems in Klebsiella and other Enterobacterales 2 (CRACKLE-2) study. Hybrid short-read and long-read genome assemblies were generated from 119 CR Kp isolates (95 originated from Houston hospitals). A comprehensive characterization of phylogenies, gene transfer, and plasmid content with pan-genome analysis was performed on all CR Kp isolates. Plasmid mating experiments were performed with CG307 and CG258 isolates of interest. Dissection of the accessory genomes suggested independent evolution and limited horizontal gene transfer between CG307 and CG258 lineages. CG307 contained a diverse repertoire of mobile genetic elements, which were shared with other non-CG258 K. pneumoniae isolates. Three unique clades of Houston CG307 isolates clustered distinctly from other global CG307 isolates, indicating potential selective adaptation of particular CG307 lineages to their respective geographical niches. CG307 strains were often isolated from the urine of hospitalized patients, likely serving as important reservoirs for genes encoding carbapenemases and extended-spectrum β-lactamases. Our findings suggest parallel cocirculation of high-risk lineages with potentially divergent evolution. IMPORTANCE The prevalence of carbapenem-resistant Klebsiella pneumoniae (CR Kp ) infections in nosocomial settings remains a public health challenge. High-risk clones such as clonal group 258 (CG258) are particularly concerning due to their association with bla KPC carriage, which can severely complicate antimicrobial treatments. There is a recent emergence of clonal group 307 (CG307) worldwide with little understanding of how this successful clone has been able to adapt while cocirculating with CG258. We provide the first evidence of potentially divergent evolution between CG258 and CG307 with limited sharing of adaptive genes. Houston, TX, is home to the largest medical center in the world, with a large influx of domestic and international patients. Thus, our unique geographical setting, where two pandemic strains of CR Kp are circulating, provides an indication of how differential accessory genome content can drive stable, endemic populations of CR Kp . Pan-genomic analyses such as these can reveal unique signatures of successful CR Kp dissemination, such as the CG307-associated plasmid (pCG307_HTX), and provide invaluable insights into the surveillance of local carbapenem-resistant Enterobacterales (CRE) epidemiology.