Autochthonous Plasmodium vivax Infections, Florida, USA, 2023.

During May-July 2023, a cluster of 7 patients at local hospitals in Florida, USA, received a diagnosis of Plasmodium vivax malaria. Whole-genome sequencing of the organism from 4 patients and phylogenetic analysis with worldwide representative P. vivax genomes indicated probable single parasite introduction from Central/South America.

Detection of resistance to macrolides and fluoroquinolones in Mycoplasma genitalium by targeted next-generation sequencing.

Mycoplasma genitalium is fastidious to culture, and its detection in human clinical specimens relies mainly on molecular methods. Phenotypic determination of antibiotic susceptibility for this bacterium is not a timely or feasible option for most clinical laboratories. This study sought to determine whether next-generation sequencing technologies can effectively be employed in determining genetic mutations associated with drug resistance in M. genitalium samples collected in Aptima Hologic tubes and possibly integrating them into viable workflows in public health laboratories. Following analysis by a custom-designed bioinformatics pipeline, at least one mutation/sample has been identified in 94/98 specimens in at least one of seven loci (macrolides: rrl, rplD, rplV; fluoroquinolones: parC, parE, gyrA, gyrB) described previously to be connected to antibiotic resistance. This method identified a total of 469 single nucleotide polymorphisms (SNPs) (452 mutations): 134 of 23S rRNA SNPs and 318 amino acid mutations: 114 substitutions and 204 synonymous; the turnaround time (sample to analyzed sequence) was typically 3 days. The assays and workflows described in this work demonstrated that the determination of a drug resistance profile for macrolides and fluoroquinolones of M. genitalium samples by using next-generation sequencing in clinical samples is a feasible approach that can be implemented in clinical laboratories, following thorough and extensive validation studies.IMPORTANCEThe mechanisms of drug resistance in Mycoplasma genitalium are complex and involve several genetic loci. The molecular methods for accurately characterizing resistance to fluoroquinolones and macrolides in this organism are often not available or approved for patient use and do not cover all genetic determinants. To this end, we propose a next-generation sequencing-based method with a turnaround time of 3 days that includes the investigation of all drug resistance loci of M. genitalium. Following adaptation, validation, and verification for routine clinical use, assays based on this method may yield molecular results that can be used to guide proper treatment regimens and for surveillance of drug resistance in the general population.

Partnerships Involved in Public Health Testing for Zika Virus in Florida, 2016.

The emergence of Zika virus in the Americas in 2015 and its association with birth defects and other adverse health outcomes triggered an unprecedented public health response and a demand for testing. In 2016, when Florida exceeded state public health laboratory capacity for diagnostic testing, the state formed partnerships with federal and commercial laboratories. Eighty-two percent of the testing (n = 33 802 of 41 008 specimens) by the laboratory partners, including Florida's Bureau of Public Health Laboratories (BPHL; n = 13 074), a commercial laboratory (n = 19 214), and the Centers for Disease Control and Prevention (CDC; n = 1514), occurred from July through November 2016, encompassing the peak period of local transmission. These partnerships allowed BPHL to maintain acceptable test turnaround times of 1 to 4 days for nucleic acid testing and 3 to 7 days for serologic testing. Lessons learned from this response to inform future outbreaks included the need for early planning to establish outside partnerships, adding specimen triage strategies to surge plans, and integrating state and CDC information systems.

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.

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.

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.

First confirmed cases of Middle East respiratory syndrome coronavirus (MERS-CoV) infection in the United States, updated information on the epidemiology of MERS-CoV infection, and guidance for the public, clinicians, and public health authorities - May 2014.

Since mid-March 2014, the frequency with which cases of Middle East respiratory syndrome coronavirus (MERS-CoV) infection have been reported has increased, with the majority of recent cases reported from Saudi Arabia and United Arab Emirates (UAE). In addition, the frequency with which travel-associated MERS cases have been reported and the number of countries that have reported them to the World Health Organization (WHO) have also increased. The first case of MERS in the United States, identified in a traveler recently returned from Saudi Arabia, was reported to CDC by the Indiana State Department of Health on May 1, 2014, and confirmed by CDC on May 2. A second imported case of MERS in the United States, identified in a traveler from Saudi Arabia having no connection with the first case, was reported to CDC by the Florida Department of Health on May 11, 2014. The purpose of this report is to alert clinicians, health officials, and others to increase awareness of the need to consider MERS-CoV infection in persons who have recently traveled from countries in or near the Arabian Peninsula. This report summarizes recent epidemiologic information, provides preliminary descriptions of the cases reported from Indiana and Florida, and updates CDC guidance about patient evaluation, home care and isolation, specimen collection, and travel as of May 13, 2014.

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.

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.

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.

An accelerated method for isolation of Salmonella enterica serotype Typhimurium from artificially contaminated foods, using a short preenrichment, immunomagnetic separation, and xylose-lysine-desoxycholate agar (6IX method).

Rapid isolation of Salmonella from food is essential for faster typing and source tracking in an outbreak. The objective of this study was to investigate a rapid isolation method that would augment the standard U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) method. Food samples with low microbial load, including egg salad and ice cream, moderately high-microbial-load tomatoes, and high-microbial-load ground beef were intentionally inoculated with 2 to 48 CFU of Salmonella enterica serotype Typhimurium. The samples were preenriched in buffered peptone water for 6 h, and then selectively concentrated by immunomagnetic separation and plated for isolation on xylose-lysine-desoxycholate agar: the 6IX method. Salmonella Typhimurium was presumptively identified from approximately 97% of the low-microbial-load and moderately high-microbial-load samples by the 6IX method 2 days before the BAM standard method for isolation of Salmonella. In 49% of the beef samples, Salmonella Typhimurium was presumptively identified 1 or 2 days earlier by the 6IX method. Given the inocula used, our data clearly indicated that for most of the food samples tested, with the exception of ground beef, Salmonella Typhimurium could be isolated two laboratory days earlier with the 6IX method compared with the BAM method. In conclusion, this 6IX method may expedite Salmonella isolation and, therefore, has the potential to accelerate strain tracking for epidemiological analysis in a foodborne outbreak.

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.

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)).

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 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.

Comparison of antibiotic susceptibility profiles and molecular typing patterns of clinical and environmental Salmonella enterica serotype Newport.

The genus Salmonella is composed of more than 2,400 serotypes, many of which cause enteric diseases in humans and animals. Several Salmonella serotypes are multidrug resistant, and there is evidence of the clonal spread of these strains from animals to humans. Salmonella enterica serotype Newport is one of the serotypes that increasingly present a multidrug-resistant phenotype. Source tracking and antibiotic resistance testing are important considerations for identifying the outbreak strain. The first goal of this study was to examine the antibiotic susceptibility patterns of clinical and environmental Salmonella Newport isolates from various geographic locations and to compare the discriminatory ability of two DNA fingerprinting techniques. The second goal was to determine whether the antibiotic resistance profiles and typing patterns correlated. Thirty Salmonella Newport isolates, including environmental and human clinical strains, were subjected to pulsed-field gel electrophoresis (PFGE), ribotyping, and antibiotic susceptibility testing. Eighty percent of the isolates showed total or intermediate resistance to one or more drugs; 75% of the isolates were multidrug resistant. Ribotyping with the EcoRI enzyme and PFGE with the XbaI enzyme each divided the isolates into 14 groups. Cluster analysis based on antibiotic susceptibility patterns generated 23 profiles. The susceptible and resistant isolates were not differentiated on the basis of either of the molecular typing techniques. Hence, no correlation was observed between the antibiotic resistance profiles and the DNA subtyping patterns. In conclusion, ribotyping is as discriminatory as PFGE and, when used in combination with antibiotic resistance profiles, provides a powerful tool for the source tracking of Salmonella Newport.