ROCker Models for Reliable Detection and Typing of Short-Read Sequences Carrying beta-Lactamase Genes

Identification of genes encoding beta-lactamases (BLs) from short-read sequences remains challenging due to the high frequency of shared amino acid functional domains and motifs in proteins encoded by BL genes and related non-BL gene sequences. Divergent BL homologs can be frequently missed during similarity searches, which has important practical consequences for monitoring antibiotic resistance. To address this limitation, we built ROCker models that targeted broad classes (e.g., class A, B, C, and D) and individual families (e.g., TEM) of BLs and challenged them with mock 150-bp- and 250-bp-read data sets of known composition. ROCker identifies most-discriminant bit score thresholds in sliding windows along the sequence of the target protein sequence and hence can account for nondiscriminative domains shared by unrelated proteins. BL ROCker models showed a 0% false-positive rate (FPR), a 0% to 4% false-negative rate (FNR), and an up-to-50-fold-higher F1 score [2 x precision x recall/(precision + recall)] compared to alternative methods, such as similarity searches using BLASTx with various e-value thresholds and BL hidden Markov models, or tools like DeepARG, ShortBRED, and AMRFinder. The ROCker models and the underlying protein sequence reference data sets and phylogenetic trees for read placement are freely available through http://enve-omics.ce.gatech.edu/data/rocker-bla. Application of these BL ROCker models to metagenomics, metatranscriptomics, and high-throughput PCR gene amplicon data should facilitate the reliable detection and quantification of BL variants encoded by environmental or clinical isolates and microbiomes and more accurate assessment of the associated public health risk, compared to the current practice. IMPORTANCE Resistance genes encoding beta-lactamases (BLs) confer resistance to the widely prescribed antibiotic class beta-lactams. Therefore, it is important to assess the prevalence of BL genes in clinical or environmental samples for monitoring the spreading of these genes into pathogens and estimating public health risk. However, detecting BLs in short-read sequence data is technically challenging. Our ROCker model-based bioinformatics approach showcases the reliable detection and typing of BLs in complex data sets and thus contributes toward solving an important problem in antibiotic resistance surveillance. The ROCker models developed substantially expand the toolbox for monitoring antibiotic resistance in clinical or environmental settings

Climatic oscillations during the Precambrian era

A remarkably regular cyclicity with a fundamental period of ~11–12 cycles is preserved in the 680 million year old Elatina formation of South Australia. All but one of the many periods present can be interpreted as resulting from the combined influences of the sunspot cycle and the lunar nodal tide - in particular, beating between these two cycles gives rise to a long period phase alternation. Available paleontological evidence is used to constrain the lunar distance 680 Ma ago, thereby constraining the length of the lunar nodal tide. We then infer from the beat period that the sunspot cycle was 10.8 ± 0.2 years, which is in agreement with independent astronomical evidence suggesting that the sunspot cycle would then have been some 3–10% shorter than it is at present. Although this interpretation is not consistent with the 12.0 year sunspot cycle counted by Williams and Sonett (1985), we demonstrate that unavoidable random errors made in discriminating unusually indistinct varves gives rise to a systematic overcount of varves of this magnitude. The clarity of this evidence for solar and lunar signals in the climate 680 Ma ago lends support to reports of their importance today.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42576/1/10584_2004_Article_BF00143906.pd

Characterization of the bacterial communities of life stages of free living lone star ticks (Amblyomma americanum).

The lone star tick (Amblyomma americanum) is an abundant and aggressive biter of humans, domestic animals, and wildlife in the southeastern-central USA and an important vector of several known and suspected zoonotic bacterial pathogens. However, the biological drivers of bacterial community variation in this tick are still poorly defined. Knowing the community context in which tick-borne bacterial pathogens exist and evolve is required to fully understand the ecology and immunobiology of the ticks and to design effective public health and veterinary interventions. We performed a metagenomic survey of the bacterial communities of questing A. americanum and tested 131 individuals (66 nymphs, 24 males, and 41 females) from five sites in three states. Pyrosequencing was performed with barcoded eubacterial primers targeting variable 16S rRNA gene regions 5-3. The bacterial communities were dominated by Rickettsia (likely R. amblyommii) and an obligate Coxiella symbiont, together accounting for 6.7-100% of sequences per tick. DNAs from Midichloria, Borrelia, Wolbachia, Ehrlichia, Pseudomonas, or unidentified Bacillales, Enterobacteriaceae, or Rhizobiales groups were also detected frequently. Wolbachia and Midichloria significantly co-occurred in Georgia (p<0.00001), but not in other states. The significance of the Midichloria-Wolbachia co-occurrence is unknown. Among ticks collected in Georgia, nymphs differed from adults in both the composition (p = 0.002) and structure (p = 0.002) of their bacterial communities. Adults differed only in their community structure (p = 0.002) with males containing more Rickettsia and females containing more Coxiella. Comparisons among adult ticks collected in New York and North Carolina supported the findings from the Georgia collection despite differences in geography, collection date, and sample handling, implying that the differences detected are consistent attributes. The data also suggest that some members of the bacterial community change during the tick life cycle and that some sex-specific attributes may be detectable in nymphs

\u3cem\u3eRickettsia typhi\u3c/em\u3e Is Still Present in Southeastern Georgia, USA

Background: Rickettsia typhi is the causative agent of murine typhus, a zoonotic disease that is sustained in nature primarily through the rat to rat flea transmission cycle. Southeastern Georgia was historically associated with outbreaks of this disease; however, its current occurrence in Georgia is unknown. Rickettsia felis, another human pathogen, is primarily associated with cat fleas and can often be found on cats, dogs, and opossums. The purpose of this study was to characterize rickettsial bacteria that are associated with fleas infesting companion and wild animals in Southeastern Georgia. Methods: Fleas were collected from companion cats, dogs, and from opossums and a cotton rat that were trapped in woodland nonhuman inhabited areas in Bulloch, Fulton and Warren Counties, Georgia in 2011-2014. The fleas were identified to species and the DNA was extracted using Qiagen DNeasy kit according to the manufacturer’s directions. Flea DNA were individually tested using TaqMan assays specific for R. typhi and R. felis. Results: Only Ctenocephalides felis were collected from cats. Dogs were infested with C. felis and Pulex simulans (n=2). Opossums were mainly infested with Polygenis gwyni (n=68) while only a few C. felis (7) and Orchopeas howardi (3), were found. R. typhi DNA was detected in 4% of P. gwyni and 12% of C. felis tested, R. felis DNA was detected in 76.61% of C. felis and 14% of P. gwyni tested. Summary and Conclusions: We demonstrated that R. typhi is still present in the area and that there is a prevalent circulation of R. felis in C. felis in Georgia. This is the first report of this agent in P. gwyni. Since R. typhi is still present it may be associated with other reservoir hosts, outside of its classic urban ratrat flea cycle. Our data suggests that companion animals may become infested when coming in contact with sylvatic animals and vice versa

Life stage and geographic origin of <i>Amblyomma americanum</i> ticks used in bacterial community analyses.

§<p>Nymphs collected in July 2010; adults in July 2010 and May 2011.</p><p>*Nymphs collected in August 2010; adults in May 2011.</p><p>NT = Not tested.</p

Multivariate analysis of the effect of sex and life stage on the relatedness of <i>Amblyomma americanum</i> bacterial communities.

<p>Tick DNAs from the archival collection (n = 27) were older, collected in a different geographical region, and processed using different protocols than tick DNAs from the Georgia collection (n = 104).</p><p>*Corrected for multiple comparisons using Holm’s method; α  = 0.05.</p