Travel Surveillance and Genomics Uncover a Hidden Zika Outbreak during the Waning Epidemic.

The Zika epidemic in the Americas has challenged surveillance and control. As the epidemic appears to be waning, it is unclear whether transmission is still ongoing, which is exacerbated by discrepancies in reporting. To uncover locations with lingering outbreaks, we investigated travel-associated Zika cases to identify transmission not captured by reporting. We uncovered an unreported outbreak in Cuba during 2017, a year after peak transmission in neighboring islands. By sequencing Zika virus, we show that the establishment of the virus was delayed by a year and that the ensuing outbreak was sparked by long-lived lineages of Zika virus from other Caribbean islands. Our data suggest that, although mosquito control in Cuba may initially have been effective at mitigating Zika virus transmission, such measures need to be maintained to be effective. Our study highlights how Zika virus may still be "silently" spreading and provides a framework for understanding outbreak dynamics. VIDEO ABSTRACT.

Genomic epidemiology reveals multiple introductions of Zika virus into the United States.

Zika virus (ZIKV) is causing an unprecedented epidemic linked to severe congenital abnormalities. In July 2016, mosquito-borne ZIKV transmission was reported in the continental United States; since then, hundreds of locally acquired infections have been reported in Florida. To gain insights into the timing, source, and likely route(s) of ZIKV introduction, we tracked the virus from its first detection in Florida by sequencing ZIKV genomes from infected patients and Aedes aegypti mosquitoes. We show that at least 4 introductions, but potentially as many as 40, contributed to the outbreak in Florida and that local transmission is likely to have started in the spring of 2016-several months before its initial detection. By analysing surveillance and genetic data, we show that ZIKV moved among transmission zones in Miami. Our analyses show that most introductions were linked to the Caribbean, a finding corroborated by the high incidence rates and traffic volumes from the region into the Miami area. Our study provides an understanding of how ZIKV initiates transmission in new regions.

Zika virus evolution and spread in the Americas.

Although the recent Zika virus (ZIKV) epidemic in the Americas and its link to birth defects have attracted a great deal of attention, much remains unknown about ZIKV disease epidemiology and ZIKV evolution, in part owing to a lack of genomic data. Here we address this gap in knowledge by using multiple sequencing approaches to generate 110 ZIKV genomes from clinical and mosquito samples from 10 countries and territories, greatly expanding the observed viral genetic diversity from this outbreak. We analysed the timing and patterns of introductions into distinct geographic regions; our phylogenetic evidence suggests rapid expansion of the outbreak in Brazil and multiple introductions of outbreak strains into Puerto Rico, Honduras, Colombia, other Caribbean islands, and the continental United States. We find that ZIKV circulated undetected in multiple regions for many months before the first locally transmitted cases were confirmed, highlighting the importance of surveillance of viral infections. We identify mutations with possible functional implications for ZIKV biology and pathogenesis, as well as those that might be relevant to the effectiveness of diagnostic tests.

Susceptibility of 169 USA300 methicillin-resistant Staphylococcus aureus isolates to two copper-based biocides, CuAL42 and CuWB50.

OBJECTIVES: To test the activity of two copper-based biocides, CuAL42 and CuWB50, and benzalkonium chloride against 169 isolates of methicillin-resistant Staphylococcus aureus (MRSA) pulsotype USA300, a virulent, multiply resistant, widespread clone in the USA. METHODS: Tests including MIC, MBC and time-kill studies were performed multiple times. RESULTS: The MIC range, MIC(50) and MIC(90) (0.59-18.75, 4.69 and 4.69 ppm, respectively) and the MBC range, MBC(50) and MBC(90) (1.17-18.75, 4.69 and 9.38 ppm, respectively) for CuAL42 were identical with those obtained with CuWB50, except that the MBC range for CuWB50 was wider (0.59-37.5 ppm). In time-kill studies, a 6 log(10) reduction of cfu was achieved within 1 h (150 ppm) and 0.5 h (300 ppm) for CuAL42, and 1.5 h (150 ppm) and 0.75 h (300 ppm) for CuWB50. CONCLUSIONS: Both copper-based biocides can effectively kill USA300 MRSA and may facilitate the eradication of the organism from healthcare settings.

Complex Epidemiological Dynamics of Eastern Equine Encephalitis Virus in Florida.

Eastern equine encephalitis virus (EEEV) infection results in high mortality in infected horses and humans. Florida has been identified as an important source of EEEV epidemics to other states in the United States. In this study, we further characterized the epidemiological and evolutionary dynamics of EEEV in Florida. Epidemiological analysis of sentinel chicken seroconversion rates to EEEV infections during 2005-2016 suggested significant seasonality of EEEV activity in Florida. We observed significant annual activity of EEEV in the North and North Central regions, with little significant seasonality in the Panhandle region. Phylogenetic analysis of complete EEEV genome sequences from different host sources and regions in Florida during 1986-2014 revealed extensive genetic diversity and spatial dispersal of the virus within Florida and relatively more clustering of the viruses in the Panhandle region. We found no significant association between EEEV genetic variation and host source. Overall, our study revealed a complex epidemiological dynamic of EEEV within Florida, implicating the Panhandle region as a possible source of the virus with sustained year-round transmission. These findings will help in implementing targeted control measures that can have the most impact in reducing or eliminating EEEV and other mosquito-borne viral infections within Florida and in the rest of the United States.

Two-year study evaluating the potential loss of methicillin resistance in a methicillin-resistant Staphylococcus aureus culture collection.

A reported loss of mecA prompted us to monitor 360 cryostocked methicillin-resistant Staphylococcus aureus strains for stability. Concurrently, 14 well-characterized strains were stored in a Microbank preservation system and subjected to multiple freeze-thaw events. There were no significant declines in the methicillin-resistant populations with either method over a two-year period.

Molecular characterization of Vibrio parahaemolyticus strains associated with foodborne illness in Florida.

Vibrio parahaemolyticus is the leading cause of bacterial seafood-based illness in the United States. Real-time PCR, pandemic group-specific PCR, ribotyping, and multilocus sequence typing were used to characterize 30 strains of V. parahaemolyticus including 11 strains associated with foodborne outbreaks in Florida and 6 known pandemic strains. Thirteen strains were positive for four pandemic group-specific PCR markers, including 5 strains associated with outbreaks in Florida. Molecular typing methods were used to further define the pandemic status of these strains because the current PCR markers are not sufficient to identify pandemic isolates. Nine of the Florida strains clustered with a majority of the known pandemic strains, based on ribotyping patterns using PvuII, but no isolated pandemic branch was formed. Using multilocus sequence typing, it was determined that 14 strains possess a previously determined pandemic sequence type. This study identified 13 novel sequence types and seven to nine novel alleles for each locus. Furthermore, the results indicate that seven of the strains from recent foodborne outbreaks in Florida are pandemic strains, and that multilocus sequence typing was the most accurate molecular method to identify these strains.

Virtual digest identification of secondary enzymes for use in pulsed-field gel electrophoresis of Staphylococcus aureus.

Pulsed-field gel electrophoresis (PFGE) is currently the gold standard for methicillin-resistant Staphylococcus aureus (MRSA) typing but only one enzyme, SmaI, is currently used for restriction digest. We report the use of virtual digestion to identify enzymes for S. aureus PFGE. Two enzymes (EagI and SacII) were identified and successfully used to characterize two sets of S. aureus isolates, 12 USA300, and 14 additional MRSA isolates comprised of seven SmaI patterns. Phylogenetic analysis of patterns generated by all enzymes determined that the USA300 MRSAs are identical. In contrast, digestion with EagI or SacII resolved one to two band differences among three MRSA pattern sets that were not detected using SmaI. These results demonstrate that a second enzyme may detect differences in S. aureus isolates not detected by single enzyme digestion. However, because isolates differing by one to two bands are considered identical, such discrimination may not be clinically or epidemiologically relevant.

Real-time PCR detection of the thermostable direct hemolysin and thermolabile hemolysin genes in a Vibrio parahaemolyticus cultured from mussels and mussel homogenate associated with a foodborne outbreak.

Molecular methods have become vital epidemiological tools in the detection and characterization of bacteria associated with a foodborne outbreak. We used both culture and real-time PCR to detect a Vibrio parahaemolyticus isolate associated with a foodborne outbreak. The outbreak occurred in July 2002 in Polk County, Florida, and originated at a Chinese buffet, with one person being hospitalized. The hospital isolated V. parahaemolyticus from the patient but destroyed the sample after diagnosis. From an onsite visit of the restaurant, food samples that possibly contributed to the outbreak were collected and sent to the Florida Department of Health, Tampa Branch Laboratory. Crab legs, crabsticks, and mussel samples were homogenized and incubated according to the Food and Drug Administration Bacteriological Analytical Manual culture protocol. Three sets of primers and a TaqMan probe were designed to target the tdh, trh, and tlh genes and used for real-time PCR. This study was successful in isolating V. parahaemolyticus from a mussel sample and detecting two of its genes (tdh and tlh) in food and pure culture by real-time PCR.

Draft Genome Sequence of Strain CBD-635, a Methicillin-Resistant Staphylococcus aureus USA100 Isolate.

We present the draft genome sequence of methicillin-resistant Staphylococcus aureus strain CBD-635, from the USA100 lineage. This is a sepsis isolate obtained from Tampa General Hospital. This strain is spa type t003 and multilocus sequence typing (MLST) type ST5, and it has been used by our group in the study of novel antimicrobial chemotherapeutics.

Bacillus Strains Most Closely Related to Bacillus nealsonii Are Not Effectively Circumscribed within the Taxonomic Species Definition.

Bacillus strains with >99.7% 16S rRNA gene sequence similarity were characterized with DNA:DNA hybridization, cellular fatty acid (CFA) analysis, and testing of 100 phenotypic traits. When paired with the most closely related type strain, percent DNA:DNA similarities (% S) for six Bacillus strains were all far below the recommended 70% threshold value for species circumscription with Bacillus nealsonii. An apparent genomic group of four Bacillus strain pairings with 94%-70% S was contradicted by the failure of the strains to cluster in CFA- and phenotype-based dendrograms as well as by their differentiation with 9-13 species level discriminators such as nitrate reduction, temperature range, and acid production from carbohydrates. The novel Bacillus strains were monophyletic and very closely related based on 16S rRNA gene sequence. Coherent genomic groups were not however supported by similarly organized phenotypic clusters. Therefore, the strains were not effectively circumscribed within the taxonomic species definition.

Improvement of a selective media for the isolation of B. anthracis from soils.

To prove linkage between an environmental sample and an anthrax case, there must be isolates obtained from both that can be compared. Although Bacillus anthracis is easily isolated from powder samples, isolating it from soil is difficult because of the high bacterial count in it. Formulations of PLET were prepared, inoculated with B. anthracis, B. cereus and B. thuringiensis and examined for growth. Two hundred eighty-three isolates including 23 B. anthracis were placed onto one formulation while MICs against trimethoprim-sulfamethoxazole were determined. The media supported B. anthracis growth at 30 degrees C and inhibited almost all other bacterial growth, including closely-related species. Sensitivity for B. anthracis and selectivity against other Bacillus and against non-Bacillus were 96.8%, 100% and 97.2% respectively. Isolates that grew had MICs >4 and >76 microg mL(-1) against trimethoprim and sulfamethoxazole, respectively. Soils spiked with 10(2)B. anthracis spores and suspended in PLET broth yielded a 6-7 log(10) increase in B. anthracis. Other growth was inhibited. PLET supplemented with sulfamethoxazole (38 microg mL(-1)), trimethoprim (2 microg mL(-1)), polymyxin B (15,000 U L(-1)), and lysozyme (150,000 U L(-1)) can successfully select for B. anthracis and will facilitate agricultural, environmental and forensic investigations of B. anthracis isolates.

Susceptibility of Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides and Bacillus thuringiensis to 24 antimicrobials using Sensititre automated microbroth dilution and Etest agar gradient diffusion methods.

OBJECTIVES: To examine susceptibilities of Bacillus anthracis and related species to 24 antimicrobials using and concurrently comparing two methods. METHODS: Twenty-four antimicrobials were tested against 95 isolates of the Bacillus cereus group including 18 B. anthracis, 42 B. cereus, 5 Bacillus mycoides, 5 Bacillus mycoides/pseudomycoides, 6 Bacillus pseudomycoides and 19 Bacillus thuringiensis to determine their MICs, MIC ranges, MIC50s and MIC90s with Etest and Sensititre at 30 and 35 degrees C for 18, 24 and 48 h. RESULTS: Both methods yielded near-identical results at both temperatures for all antimicrobials except trimethoprim/sulfamethoxazole. Resistance to trimethoprim/sulfamethoxazole in 97% (92/95) was not always evident until tests were incubated for 48 h at 30 degrees C. All B. anthracis isolates were susceptible to 22 antimicrobials and resistant to trimethoprim/sulfamethoxazole while three isolates were erythromycin-intermediate. Whereas the B. thuringiensis were resistant to the beta-lactams, two B. cereus, one B. mycoides, five B. pseudomycoides and two B. mycoides/pseudomycoides were susceptible. Three B. cereus were solely clindamycin-resistant. Of the seven erythromycin-intermediate or -resistant B. cereus, three were resistant to clindamycin and one was resistant to clarithromycin and clindamycin. One B. mycoides was intermediately resistant to quinupristin/dalfopristin and meropenem and one was clindamycin-resistant. All B. pseudomycoides were clindamycin-resistant with one quinupristin/dalfopristin-resistant. Two B. mycoides/pseudomycoides were intermediately resistant to quinupristin/dalfopristin and clindamycin and a third was intermediately resistant to clindamycin alone. All isolates were susceptible to chloramphenicol, ciprofloxacin, gatifloxacin, gentamicin, levofloxacin, linezolid, moxifloxacin, rifampicin, streptomycin, tetracycline, tigecycline and vancomycin. CONCLUSIONS: This paper expands the list of therapeutic or prophylactic antimicrobials potentially effective against B. cereus group isolates using two testing methods that produced comparable results.

Bacillus acidiceler sp. nov., isolated from a forensic specimen, containing Bacillus anthracis pX02 genes.

Research at the Center for Biological Defense identified plasmid-borne forms of Bacillus anthracis pXO2 genes in a Gram-positive, endospore-forming rod, isolated from a forensic specimen considered a credible threat of harbouring anthrax. Conventional, commercial and molecular-based methods indicated that the isolate (CBD 119(T)) was not B. anthracis and considered not to be a member of the Bacillus cereus group. Based on the 16S rRNA gene sequence similarities, strain CBD 119(T) was most closely related to Bacillus luciferensis LMG 18422(T) (99.3 %). Phenotyping and fatty acid methyl ester analysis of the isolate were conducted alongside B. luciferensis JCM 12212(T). The major cellular fatty acids (anteiso-C(15 : 0), iso-C(15 : 0), and >7 iso or anteiso forms) supported inclusion of the isolate in the genus Bacillus. Strain CBD 119(T) was inconsistent with B. luciferensis JCM 12212(T) for 18 of 96 traits evaluated including motility, degree of endospore-driven swelling and pH optimum; the two were linked by fatty acid methyl ester analysis as separate but closely related species. DNA-DNA relatedness between strain CBD 119(T) and B. luciferensis JCM 12212(T) resulted in less than 20 % hybridization. The results of biochemical and physiological characterization, chemotaxonomic analysis and DNA-DNA hybridization differentiated strain CBD 119(T) both phenotypically and genotypically from the only species with validly published name with greater than 97 % 16S rRNA gene sequence similarity. The isolate has an accelerated doubling time when grown in aerated broth at pH 5.9 relative to that at pH 7.1. Therefore, it is proposed that strain CBD 119(T) represents a novel species, Bacillus acidiceler sp. nov. The type strain is strain CBD 119(T) (=NRRL B-41736(T)=DSM 18954(T)).

Community-associated methicillin-resistant Staphylococcus aureus epidemic clone USA300 in isolates from Florida and Washington.

We examined 299 methicillin-resistant, community-associated Staphylococcus aureus isolates from Florida and Washington State for the presence of the USA300 epidemic clone. Pulsed-field gel electrophoresis demonstrated the epidemic clone in 43% of our S. aureus strains and in isolates from both states. The majority of the USA300 isolates (88%) were from wound infections.

Bacillus anthracis virulent plasmid pX02 genes found in large plasmids of two other Bacillus species.

In order to cause the disease anthrax, Bacillus anthracis requires two plasmids, pX01 and pX02, which carry toxin and capsule genes, respectively, that are used as genetic targets in the laboratory detection of the bacterium. Clinical, forensic, and environmental samples that test positive by PCR protocols established by the Centers for Disease Control and Prevention for B. anthracis are considered to be potentially B. anthracis until confirmed by culture and a secondary battery of tests. We report the presence of 10 genes (acpA, capA, capB, capC, capR, capD, IS1627, ORF 48, ORF 61, and repA) and the sequence for the capsule promoter normally found on pX02 in Bacillus circulans and a Bacillus species closely related to Bacillus luciferensis. Tests revealed these sequences to be present on a large plasmid in each isolate. The 11 sequences consistently matched to B. anthracis plasmid pX02, GenBank accession numbers AF188935.1, AE011191.1, and AE017335.3. The percent nucleotide identities for capD and the capsule promoter were 99.9% and 99.7%, respectively, and for the remaining nine genes, the nucleotide identity was 100% for both isolates. The presence of these genes, which are usually associated with the pX02 plasmid, in two soil Bacillus species unrelated to B. anthracis alerts us to the necessity of identifying additional sequences that will signal the presence of B. anthracis in clinical, forensic, and environmental samples.

Use of two selective media and a broth motility test can aid in identification or exclusion of Bacillus anthracis.

During the anthrax attack of 2001, the Florida Department of Health (FDOH) Bureau of Laboratories in Tampa received hundreds of isolates suspected of being Bacillus anthracis. None were confirmed to be B. anthracis since most isolates were motile and not even in the Bacillus cereus group. Although the sentinel laboratories now send fewer isolates to FDOH laboratories, should another attack occur the number of isolates submitted would likely increase dramatically, and this upsurge would seriously challenge personnel who are expected to be busy examining an increased number of environmental samples. We examined two selective and differential growth media and alternative motility methods that could be used to streamline the processing of suspicious isolates. Of 60 isolates previously sent to the FDOH laboratory, 56 were endospore-forming gram-positive rods and only 7 grew on mannitol-egg yolk-polymyxin B agar and/or the Anthracis chromogenic agar. Microscopic observation of early-log-phase growth (2 to 3 h) in a shaking broth was the best method to detect motility in 40 isolates that appeared nonmotile in the motility media investigated. One of these growth media and microscopic examination of shaken broth cultures can be used to show that an isolate is not B. anthracis before expensive molecular and antibody-based tests are performed. By doing so, costs could be reduced and analysis time shortened.

The stability of the Chlorella nitrate reductase mRNA is determined by the secondary structure of the 5'-UTR: implications for posttranscriptional regulation of nitrate reductase.

Nitrate reductase (NR; EC 1.6.1.1-3) can be controlled at both transcriptional and posttranscriptional levels. Here we describe stability of NR mRNA as a mechanism of control. The NR gene in Chlorella vulgaris (Warburg strain) transcribes a stable mRNA and an unstable mRNA. In-vitro-synthesized transcripts representing these mRNAs show the same stability characteristics. The unstable mRNA is 30 nucleotides longer at the 5'-UTR compared to the stable mRNA. Using an RNA-folding program the 5'-UTR of the longer unstable RNA showed a more extensive stem-loop structure compared to the more linear form of the shorter stable mRNA. Transcripts representing RNAs with intermediate 5'-UTRs folded similarly to the long form and were unstable, or similarly to the short form and were more stable. Thus the secondary structure of the 5'-UTR of NR mRNA is important in the stability of NR transcripts in Chlorella and allows the cell to respond to changes in nitrogen source in an energy-efficient manner.

Comparison of methods for DNA isolation from food samples for detection of Shiga toxin-producing Escherichia coli by real-time PCR.

In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.