John Gustafson

We present draft genomes of <i>Citrobacter freundii</i> strains isolated from a model wastewater treatment plant seeded with sludge from Stillwater, Oklahoma. Analysis suggests that the Stillwater strains are related to human isolates from China and the UK. All strains also possess antimicrobial resistance genes, highlighting the potential public health implications.

The Gram-negative <i>Elizabethkingia</i> express multiple antibiotic resistance and cause severe opportunistic infections. Vancomycin is commonly used to treat Gram-positive infections and has also been used to treat <i>Elizabethkingia</i> infections, even though Gram-negative organisms possess a vancomycin permeability barrier. <i>Elizabethkingia anophelis</i> appeared relatively vancomycin-susceptible and challenge with this drug led to morphological changes indicating cell lysis. In stark contrast, vancomycin growth challenge revealed that <i>E. anophelis</i> populations refractory to vancomycin emerged. In addition, <i>E. anophelis</i> vancomycin-selected mutants arose at high frequencies and demonstrated elevated vancomycin resistance and reduced susceptibility to other antimicrobials. All mutants possessed a SNP in a gene (<i>vsr1</i> = vancomycin-susceptibility regulator 1) encoding a PadR family transcriptional regulator located in the putative operon <i>vsr1-ORF551</i>, which is conserved in other <i>Elizabethkingia</i> spp as well. This is the first report linking a <i>padR</i> homologue (<i>vsr1</i>) to antimicrobial resistance in a Gram-negative organism. We provide evidence to support that <i>vsr1</i> acts as a negative regulator of <i>vsr1-ORF551</i> and that <i>vsr1-ORF551</i> upregulation is observed in vancomycin-selected mutants. Vancomycin-selected mutants also demonstrated reduced cell length indicating that cell wall synthesis is affected. ORF551 is a membrane-spanning protein with a small phage shock protein conserved domain. We hypothesize that since vancomycin-resistance is a function of membrane permeability in Gram-negative organisms, it is likely that the antimicrobial resistance mechanism in the vancomycin-selected mutants involves altered drug permeability.

Students who experience research as undergraduates tend to remain in science, technology, engineering, and mathematics fields. Since course-based undergraduate research experiences (CUREs) are often expensive to implement, we developed a relatively inexpensive first-semester research course for majors entitled “Freshman Research in Biochemistry,” in which approximately 100 students participated in an ongoing research project each year. During this course, students conducted laboratory work related to multiple research veins, and graduate teaching assistants (TAs) and the instructor ensured that sufficient data from the laboratory were made available to the students to analyze. During three unique course iterations, students worked on faculty research projects that included connecting contigs of the draft genome of a native Oklahoma bacterial Arhodomonas isolate capable of degrading petroleum hydrocarbons, cloning antibiotic resistance genes from the genus Elizabethkingia, and analyzing RNAseq data from Elizabethkingia anopheles challenged with cell wall-active antibiotics to determine gene function, gene differential expression, and relative gene genome location. During the course, students scored results, analyzed data, maintained laboratory notebooks, and presented posters in a symposium to faculty and graduate TAs. To determine positive impacts of the course on students, they completed the Laboratory Course Assessment Survey in two course sections. Survey data indicated/students agreed that the course included elements of Iteration, Collaboration, and Discovery and Relevance. Students also reported that they gained scientific skills but were ambivalent about whether instructor feedback was harsh. Examination of Student Survey of Instruction responses showed students thought positively about the course, but some considered it too difficult. Biochemistry and Molecular Biology and Biochemistry major cohort tracking revealed no effect of Freshman Research in Biochemistry on student persistence in the major or institution. Nonetheless, we consider the course a cost-conscious model for biochemistry and molecular biology departments for introducing research to first-year students.

Physiological experimentation, transcriptomics, and metabolomics were engaged to compare a fusidic acid-resistant <i>Staphylococcus aureus</i> mutant SH10001st-2 to its parent strain SH1000. SH10001st-2 harbored a mutation (H457Y) in the gene <i>fusA</i> which encodes the fusidic acid target, elongation factor G, as well as mutations in a putative phage gene of unknown function. SH10001st-2 grew slower than SH1000 at three temperatures and had reduced coagulase activity, two indicators of the fitness penalty reported for <i>fusA</i>-mediated fusidic acid- resistance in the absence of compensatory mutations. Despite the difference in growth rates, the levels of O₂ consumption and CO₂ production were comparable. Transcriptomic profiling revealed 326 genes were upregulated and 287 were downregulated in SH10001st-2 compared to SH1000. Cell envelope and transport and binding protein genes were the predominant functional categories of both upregulated and downregulated genes in SH10001st-2. Genes of virulence regulators, notably the <i>agr</i> and <i>kdp</i> systems, were highly upregulated as were genes encoding capsule production. Contrary to what is expected of mid-exponential phase cells, genes encoding secreted virulence factors were generally upregulated while those for adhesion-associated virulence factors were downregulated in SH10001st-2. Metabolomic analysis showed an overall increase in metabolite pools in SH10001st-2 compared to SH1000, mostly for amino acids and sugars. Slowed growth and metabolite accumulation may be byproducts of <i>fusA</i> mutation-mediated protein synthesis impairment, but the overall results indicate that SH10001st-2 is compensating for the H457Y fitness penalty by repurposing its virulence machinery, in conjunction with increasing metabolite uptake capacity, in order to increase nutrient acquisition.

Ciprofloxacin is a broad-spectrum antibiotic of the fluoroquinolone class that is used to treat bacterial infections in both humans and animals. Antibiotics released into the environment can select for antibiotic-resistant microorganisms, which negatively affects human and animal healthcare. Understanding soil factors that govern the mobility of ciprofloxacin can facilitate the development of targeted efforts to mitigate potential negative impacts. The objectives were (1) to determine the sorption capacity of ciprofloxacin in soils under acidic conditions; and (2) to reveal relative importance of key soil factors that influence sorption of ciprofloxacin. Evaluations were conducted using 20 soil samples with diverse properties and different cultivation/vegetation history. Sorption capacity ranged from 8 to 141 g kg −1 ; distribution coefficient ( K d ) ranged from 23 to 200 mL kg −1 soil; and soil organic carbon-water partitioning coefficient ( K oc ) values ranged from 54 to 2,146 mL g −1 organic carbon. Clay content and cation exchange capacity were the most significant factors that influenced sorption capacity and K d of ciprofloxacin with r values of 0.92 *** and 0.64 *** , respectively. Soil pH had little effect on ciprofloxacin sorption parameters with r < 0.25. pH-independent charges played a predominant role dictating sorption parameters of ciprofloxacin in soil. Cation exchange via interlayer adsorption was a primary sorption mechanism under acidic conditions. Sorption parameters were significantly correlated with organic carbon content in cultivated soils only, resulting in r values of 0.97 *** (with sorption capacity) and 0.72 *** (with K d ). Cultivation led to changes in the quality of soil organic matter, resulting in changes in the sorption behavior and altered mechanisms of ciprofloxacin sorption in soil. Soils are effective in restraining the mobility of ciprofloxacin through adsorption and the effectiveness increases with clay content.

Nicrophorus is a genus of beetles that bury and transform small vertebrate carcasses into a brood ball coated with their oral and anal secretions to prevent decay and that will serve as a food source for their young. Nicrophorus pustulatus is an unusual species with the ability to overtake brood of other burying beetles and whose secretions, unlike other Nicrophorus species, has been reported not to exhibit antimicrobial properties. This work aims to better understand how the presence or absence of a food source influences the expression of genes involved in the feeding process of N. pustulatus. To achieve that, total RNA was extracted from pooled samples of salivary gland tissue from N. pustulatus and sequenced using an Illumina platform. The resulting reads were used to assemble a de novo transcriptome using Trinity. Duplicates with more than 95% similarity were removed to obtain a "unigene" set. Annotation of the unigene set was done using the Trinotate pipeline. Transcript abundance was determined using Kallisto and differential gene expression analysis was performed using edgeR. A total of 651 genes were found to be differentially expressed, including 390 upregulated and 261 downregulated genes in fed insects compared to starved. Several genes upregulated in fed beetles are associated with the insect immune response and detoxification processes with only one transcript encoding for the antimicrobial peptide (AMP) defensin. These results confirm that N. pustulatus does not upregulate the production of genes encoding AMPs during feeding. This study provides a snapshot of the changes in gene expression in the salivary glands of N. pustulatus following feeding while providing a well described transcriptome for the further analysis of this unique burying beetle.

Comparative genomic sequencing of laboratory-derived vancomycin-intermediate <i>Staphylococcus</i><i>aureus</i> (VISA) (MM66-3 and MM66-4) revealed unique mutations in both MM66-3 (in <i>apt</i> and <i>ssaA6</i>), and MM66-4 (in <i>apt</i> and <i>walK</i>), compared to hetero-VISA parent strain MM66. Transcriptional profiling revealed that both MM66 VISA shared 79 upregulated genes and eight downregulated genes. Of these, 30.4% of the upregulated genes were associated with the cell envelope, whereas 75% of the downregulated genes were associated with virulence. In concordance with mutations and transcriptome alterations, both VISA strains demonstrated reduced autolysis, reduced growth in the presence of salt and reduced virulence factor activity. In addition to mutations in genes linked to cell wall metabolism (<i>ssaA6</i> and <i>walK</i>), the same mutation in <i>apt</i> which encodes adenine phosphoribosyltransferase, was confirmed in both MM66 VISA. Apt plays a role in both adenine metabolism and accumulation and both MM66 VISA grew better than MM66 in the presence of adenine or 2-fluoroadenine indicating a reduction in the accumulation of these growth inhibiting compounds in the VISA strains. MM66 <i>apt</i> mutants isolated via 2-fluoroadenine selection also demonstrated reduced susceptibility to the cell wall lytic dye Congo red and vancomycin. Finding that <i>apt</i> mutations contribute to reduced vancomycin susceptibility once again suggests a role for altered purine metabolism in a VISA mechanism.

In the case of a suspected anthrax attack, the United States Food and Drug Administration has a plan to immediately distribute stockpiled antibiotics to the affected population. If performed on a large scale, the amount of antibiotics entering wastewater treatment plants would quickly increase. This influx of drugs would likely be followed by unknown consequences to wastewater treatment plants, downstream ecology, and public health. We therefore investigated the influence of the antibiotic ciprofloxacin on bench scale activated sludge reactors. Reactor performance was unaffected regarding removal of total organics during 14 days of antibiotic loading; however, the reactor microbial community was affected immediately following ciprofloxacin-challenge. Aerobic colony-forming units were initially reduced at 24 h and then recovered after 72 h. Analysis of 16S rRNA gene sequences demonstrated an increased relative abundance of Burkholderiales, Bradyrhizobiaceae, Microbacteriaceae, and Emticicia spp. In addition, ciprofloxacin decreased the Shannon's diversity index and community richness. Further, analysis of randomly selected bacterial isolates demonstrated that the prevalence of multidrug resistance increased in the ciprofloxacin-challenged reactors. These results suggest that an emergency response-associated loading of ciprofloxacin into municipal wastewater treatment plants could alter the activated sludge microbiome and select for multidrug-resistant bacteria.

Tea tree oil (TTO) is hypothesized to kill bacteria by indiscriminately denaturing membrane and protein structures. A <i>Staphylococcus aureus</i> small colony variant (SCV) selected with TTO (SH1000-TTORS-1) demonstrated slowed growth, reduced susceptibility to TTO, a diminutive cell size, and a thinned cell wall. Utilizing a proteomics and metabolomics approach, we have now revealed that the TTO-selected SCV mutant demonstrated defective fatty acid synthesis, an alteration in the expression of genes and metabolites associated with central metabolism, the induction of a general stress response, and a reduction of proteins critical for active growth and translation. SH1000-TTORS-1 also demonstrated an increase in amino acid accumulation and a decrease in sugar content. The reduction in glycolytic pathway proteins and sugar levels indicated that carbon flow through glycolysis and gluconeogenesis is reduced in SH1000-TTORS-1. The increase in amino acid accumulation coincides with the reduced production of translation-specific proteins and the induction of proteins associated with the stringent response. The decrease in sugar content likely deactivates catabolite repression and the increased amino acid pool observed in SH1000-TTORS-1 represents a potential energy and carbon source which could maintain carbon flow though the tricarboxylic acid (TCA) cycle. It is noteworthy that processes that contribute to the production of the TTO targets (proteins and membrane) are reduced in SH1000-TTORS-1. This is one of a few studies describing a mechanism that bacteria utilize to withstand the action of an antiseptic which is thought to inactivate multiple cellular targets.

Although Staphylococcus aureus is a major cause of food poisoning, little is known about its response to growth on food. Utilizing a transcriptional profiling and metabolomics approach, we compared S. aureus grown on autoclaved chicken breast (ACB) to Luria broth agar. ACB cultures demonstrated increased expression of genes associated with protein synthesis, cofactors, secondary metabolites, nitrogen and nucleotide metabolism, amino acid transport, and reduced expression of general stress, lipid metabolism, and virulence genes. The ACB culture also displayed characteristics of catabolite de-repression and anaerobic growth, and increased expression of arginine biosynthesis genes (argFGH) and an arginine/ornithine antiporter gene (arcD). S. aureus synthesizes arginine from proline and the ACB culture exhibited increased expression of proline transport genes (opuBA, opuBB and putP) and increased proline accumulation. Amino acid and sugar content in the ACB grown culture increased, and this was attributed to the consumption of ACB, transport of amino acids, and gluconeogenesis. Genes involved with biotin biosynthesis and uptake were upregulated and biotin is required for amino acid catabolism. Genes encoding urease and urease activity were upregulated in ACB cultures, while urea levels were reduced. This research provides fundamental information on the response of S. aureus growing on chicken meat that could find application in future attempts to reduce the growth of S. aureus in food.

Abstract The Elizabethkingia are a genetically diverse genus of emerging pathogens that exhibit multidrug resistance to a range of common antibiotics. Two representative species, Elizabethkingia bruuniana and Elizabethkingia meningoseptica , were phenotypically tested to determine minimum inhibitory concentrations for five antibiotics. Ultra-long read sequencing with Oxford Nanopore Technologies and subsequent de novo assembly produced complete, gapless circular genomes for each strain. Alignment based annotation with Prokka identified 5,480 features in E. bruuniana and 5,203 features in E. meningoseptica , where none of these identified genes or gene combinations corresponded to observed phenotypic resistance values. Pan-genomic analysis, performed with an additional 19 Elizabethkingia strains, identified a core-genome size of 2,658,537 bp, 32 uniquely identifiable intrinsic chromosomal antibiotic resistance core-genes and 77 antibiotic resistance pan-genes. Using core-SNPs and pan-genes in combination with six machine learning algorithms, binary classification of clindamycin and vancomycin resistance achieved f1 scores of 0.94 and 0.84 respectively. Performance on the more challenging multiclass problem for fusidic acid, rifampin and ciprofloxacin resulted in f1 scores of 0.70, 0.75 and 0.54 respectively.

The <i>Elizabethkingia</i> are a genetically diverse genus of emerging pathogens that exhibit multidrug resistance to a range of common antibiotics. Two representative species, <i>Elizabethkingia bruuniana</i> and <i>E. meningoseptica</i>, were phenotypically tested to determine minimum inhibitory concentrations (MICs) for five antibiotics. Ultra-long read sequencing with Oxford Nanopore Technologies (ONT) and subsequent <i>de novo</i> assembly produced complete, gapless circular genomes for each strain. Alignment based annotation with Prokka identified 5,480 features in <i>E. bruuniana</i> and 5,203 features in <i>E. meningoseptica</i>, where none of these identified genes or gene combinations corresponded to observed phenotypic resistance values. Pan-genomic analysis, performed with an additional 19 <i>Elizabethkingia</i> strains, identified a core-genome size of 2,658,537 bp, 32 uniquely identifiable intrinsic chromosomal antibiotic resistance core-genes and 77 antibiotic resistance pan-genes. Using core-SNPs and pan-genes in combination with six machine learning (ML) algorithms, binary classification of clindamycin and vancomycin resistance achieved f1 scores of 0.94 and 0.84, respectively. Performance on the more challenging multiclass problem for fusidic acid, rifampin and ciprofloxacin resulted in f1 scores of 0.70, 0.75, and 0.54, respectively. By producing two sets of quality biological predictors, pan-genome genes and core-genome SNPs, from long-read sequence data and applying an ensemble of ML techniques, our results demonstrated that accurate phenotypic inference, at multiple AMR resolutions, can be achieved.

We report the isolation and characterization of two Elizabethkingia anophelis strains (OSUVM-1 and OSUVM-2) isolated from sources associated with horses in Oklahoma. Both strains appeared susceptible to fluoroquinolones and demonstrated high MICs to all cell wall active antimicrobials including vancomycin, along with aminoglycosides, fusidic acid, chloramphenicol, and tetracycline. Typical of the Elizabethkingia, both draft genomes contained multiple copies of β-lactamase genes as well as genes predicted to function in antimicrobial efflux. Phylogenetic analysis of the draft genomes revealed that OSUVM-1 and OSUVM-2 differ by only 6 SNPs and are in a clade with 3 strains of Elizabethkingia anophelis that were responsible for human infections. These findings therefore raise the possibility that Elizabethkingia might have the potential to move between humans and animals in a manner similar to known zoonotic pathogens.

Staphylococcus aureus small colony variants (SCV) are associated with chronic and recurring infections that are recalcitrant in antimicrobial therapy. SCVs demonstrate: slower growth rates; defective metabolism and electron transport; and reduced antimicrobial susceptibility. Tea tree oil (TTO) kills bacteria by denaturing proteins and disrupting membrane structure and TTO reduced‐susceptibility (TTO RS ) S. aureus mutants exhibit an “unique” SCV phenotype. Similar to previously described SCVs, all TTO RS SCVs investigated were less susceptible to both the cell wall antibiotics vancomycin and oxacillin. A TTO RS SCV mutant (TTO RS ‐1) harbored numerous mutations, including a mutation within acpP which encodes the acyl carrier protein (ACP) essential for fatty acid biosynthesis. Comparative proteomics revealed that TTO RS ‐1 demonstrated increases in 39 proteins and decreases in 74 proteins compared to parent strain SH1000. In TTO RS ‐1, the fatty acid biosynthesis proteins FabF (3‐oxoacyl‐synthase) and FabD (malonyl CoA‐acyl carrier protein transacylase) and the bifunctional phosphopantothenoylcysteine decarboxylase/phosphopantothenate‐cysteine ligase were found in greater abundance. This latter enzyme is required for the synthesis of 4′‐phosphopantetheine, which when linked to ACP acts as the anchor on which fatty acid biosynthesis takes place. Furthermore, RT‐PCR analysis revealed that the 5 gene fapR operon gene member plsX (encodes glycerol‐3‐phosphate acyltransferase), was up‐regulated in TTO RS ‐1. These findings indicate an alteration in fatty acid biosynthesis in TTO RS ‐1. S. aureus SCVs can result from the deletion of menB or cold shock protein cspB , and menB SCVs demonstrate a decrease in citric acid cycle activity. TTO RS ‐1 harbored less menaquinone biosynthetic protein MenB (1,4‐dihydroxy‐2‐naphthoyl‐CoA synthase), cold shock proteins CspB and CspC, ATP synthase subunit gamma, and proteins involved with the citric acid cycle. Collectively our data indicates that fatty acid biosynthesis is altered in TTO RS ‐1, as would be expected in an acpP mutant. We also demonstrate the reduced synthesis of certain proteins in TTO RS ‐1 mirroring what has been observed in previously described SCV phenotypes. Support or Funding Information Oklahoma Agricultural Experimental Station

The genus Elizabethkingia is comprised of aerobic, non‐fermenting, non‐motile and non‐spore‐forming gram‐negative rods that are widely distributed in nature. Elizabethkingia is rarely reported to cause infections in humans with only a few isolated incidents reported each year typically confined to healthcare facilities. However, a recent outbreak of an Elizabethkingia strain (sp. anophelis) occurred in Wisconsin with additional single infections in Michigan and Illinois, resulted in an unusually high morbidity (20 deaths/65 cases). Patients diagnosed with this strain of Elizabethkingia anopheles lived in unassociated areas and were present in different health facilities across the state. As of November 9, 2016, the source of this most recent outbreak is yet to be determined. Elizabethkingia is known to have multiple genes coding for antibiotic resistance, but these do not seem to have contributed to the unusual outbreak. Patients affected are exclusively immune‐suppressed with most infections occurring in the blood, leading to sepsis. One hypothesis for this change in phenotype could be due to mobile genetic elements such as transposons, integrases, plasmids and phages all of which function to facilitate DNA rearrangements. We have identified a homologous double transposon region conserved in only a few strains of Elizabethkingia suggesting a recent conjugative event. This conserved homologous region (~62,871bp) is bordered by mobile genetic elements, and found exclusively within E. anopheles NUHP‐1, PW2809, CSID_3015183678, CSID_3000521207, CSID_3015183681, CSID_3015183684, Genomospecies 2, Genomospecies 3 and E. miricola ATCC. The shortest version of this conserved region is in E. anopheles PW2809 with a slightly larger conserved region present in both Genomospecies 2 and 3. However, the largest version of this double transposon was found in E. anophelis NUHP‐1, miricola ATCC and the four anophelis CDC outbreak strains. The increased homology of this transposon in a select number of strains seems to suggest a relatively recent evolutionary event. However, the location of this highly conserved transposon (termed “Tnpx2”) changes specifically within the CDC outbreak strains the result of which causes a disruption in the gene sequence used for coding of an A/G‐specific adenine glycosylase. This potential disruption of glycolytic function along with the identification of toxin‐antitoxin complexes unique to the CDC outbreak strains may have caused a phenotypical change within the bacteria that increased its survival. We propose that the genome rearrangement, found only in the CDC outbreak strains isolated from Wisconsin, could have influenced epidemiological changes and the subsequent severity of the most recent outbreak of Elizabethkingia anophelis.

Transcriptional profiles of 2 unrelated clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were analyzed following 10% (v/v) ethanol challenge (15 min), which arrested growth but did not reduce viability. Ethanol-induced stress (EIS) resulted in differential gene expression of 1091 genes, 600 common to both strains, of which 291 were upregulated. With the exception of the downregulation of genes involved with osmotic stress functions, EIS resulted in the upregulation of genes that contribute to stress response networks, notably those altered by oxidative stress, protein quality control in general, and heat shock in particular. In addition, genes involved with transcription, translation, and nucleotide biosynthesis were downregulated. relP, which encodes a small alarmone synthetase (RelP), was highly upregulated in both MRSA strains following ethanol challenge, and relP inactivation experiments indicated that this gene contributed to EIS growth arrest. A number of persistence-associated genes were also upregulated during EIS, including those that encode toxin-antitoxin systems. Overall, transcriptional profiling indicated that the MRSA investigated responded to EIS by entering a state of dormancy and by altering the expression of elements from cross protective stress response systems in an effort to protect preexisting proteins.

Complete genome comparisons, transcriptomic and metabolomic studies were performed on two laboratory-selected, well-characterized vancomycin-intermediate Staphylococcus aureus (VISA) derived from the same parent MRSA that have changes in cell wall composition and decreased autolysis. A variety of mutations were found in the VISA, with more in strain 13136p(-)m⁺V20 (vancomycin MIC = 16 µg/mL) than strain 13136p(-)m⁺V5 (MIC = 8 µg/mL). Most of the mutations have not previously been associated with the VISA phenotype; some were associated with cell wall metabolism and many with stress responses, notably relating to DNA damage. The genomes and transcriptomes of the two VISA support the importance of gene expression regulation to the VISA phenotype. Similarities in overall transcriptomic and metabolomic data indicated that the VISA physiologic state includes elements of the stringent response, such as downregulation of protein and nucleotide synthesis, the pentose phosphate pathway and nutrient transport systems. Gene expression for secreted virulence determinants was generally downregulated, but was more variable for surface-associated virulence determinants, although capsule formation was clearly inhibited. The importance of activated stress response elements could be seen across all three analyses, as in the accumulation of osmoprotectant metabolites such as proline and glutamate. Concentrations of potential cell wall precursor amino acids and glucosamine were increased in the VISA strains. Polyamines were decreased in the VISA, which may facilitate the accrual of mutations. Overall, the studies confirm the wide variability in mutations and gene expression patterns that can lead to the VISA phenotype.

We report the draft genome of Elizabethkingia strain ATCC 33958, which has been classified as Elizabethkingia miricola. Similar to other Elizabethkingia species, the ATCC 33958 draft genome contains numerous β-lactamase genes. ATCC 33958 also harbors a urease gene cluster which supports classification as E. miricola.

This chapter outlines the impact of antimicrobial resistance by describing the epidemiology of select antimicrobial‐resistant pathogens and the difficulties associated with treatment of infections caused by these organisms. It discusses antimicrobial treatment options for infections caused by resistant enterococci, Staphylococcus aureus, Streptococcus pneumoniae, and Mycobacterium tuberculosis. The arrival of vancomycin‐resistant enterococci (VRE) is often attributed to the overuse of vancomycin, which has increased about 100‐fold in the last 20 years, predominantly to treat methicillin‐resistant S. aureus (MRSA), enterococcal, and Clostridium difficile infections. The most important risk factors for VRE colonization and infection include severe underlying disease, extended hospital stay, and previous antimicrobial exposure. In the absence of literature supporting the use of one agent versus another, linezolid should be used as first line therapy for VRE infections, because it can cover both Enterococcus faecalis and E. faecium. No population or geographical region is immune or isolated from the risk of infection with the primary cause of tuberculosis (TB) M. tuberculosis. Vaccine candidates that have been developed so far include recombinant M. bovis Bacillus Calmette‐Guérin; attenuated M. tuberculosis; subunit and pooled subunit vaccines; fusion polyproteins; and DNA vaccines.

We report the draft genome sequences of three vancomycin-susceptible methicillin-resistant Staphylococcus aureus strains. S. aureus strain MV8 is a sequence type 8 (ST-8) staphylococcal cassette chromosome mec element type IV (SCCmec IV) derivative, while the other two strains (S. aureus MM25 and MM61) are ST-5 SCCmec II strains. MM61 is also closely related to the heterogeneous vancomycin-intermediate S. aureus strain MM66.

The draft genomes of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) strain MM66 and MM66 isolates demonstrating altered vancomycin resistance levels were produced in an effort to provide information on mutations contributing to the vancomycin resistance levels observed in these strains.

Tea tree oil (TTO)-reduced susceptibility (TTORS) mutants of two Staphylococcus aureus laboratory strains were isolated utilizing TTO gradient plates. Attempts to isolate TTORS mutants employing agar plates containing single TTO concentrations failed. All TTORS mutants demonstrated a small colony variant (SCV) phenotype and produced cells with a smaller diameter, as determined by scanning electron microscopy. The addition of SCV auxotrophic supplements to media did not lead to an increase in TTORS mutant colony size. Revertants were also isolated from the TTORS mutants following growth in drug-free media, and all revertant strains demonstrated phenotypes similar to their respective parent strains. Transmission electron microscopy revealed that an SH1000 TTORS mutant demonstrated a thinner cell wall and novel septal invaginations compared with parent strain SH1000. In addition, comparative genomic sequencing did not reveal any mutations in an SH1000 TTORS mutant previously linked to well-characterized SCV genotypes. This study demonstrates that TTO can select for a unique SCV phenotype.

The rapid detection of biological contaminants, such as Escherichia coli O157:H7 and Salmonella enterica , on foods and food-processing surfaces is important to ensure food safety and streamline the food-monitoring process. Laser-induced breakdown spectroscopy (LIBS) is an ideal candidate technology for this application because sample preparation is minimal and results are available rapidly (seconds to minutes). Here, multivariate regression analysis of LIBS data is used to differentiate the live bacterial pathogens E. coli O157:H7 and S. enterica on various foods (eggshell, milk, bologna, ground beef, chicken, and lettuce) and surfaces (metal drain strainer and cutting board). The type (E. coli or S. enterica) of bacteria could be differentiated in all cases studied along with the metabolic state (viable or heat killed). This study provides data showing the potential of LIBS for the rapid identification of biological contaminants using spectra collected directly from foods and surfaces.

The primary purpose of this study was to determine if methicillin-resistant Staphylococcus aureus (MRSA) strains could be identified in the milk of dairy cattle in a Paso del Norte region dairy of the United States. Using physiological and PCR-based identification schemes, a total of 40 Staph. aureus strains were isolated from 29 raw milk samples of 133 total samples analyzed. Pulsed-field gel electrophoresis after digestion with the SmaI enzyme revealed that the 40 confirmed strains were represented by 5 pulsed-field types, which each contained 3 or more strains. Of 7 hospital strains isolated from cows undergoing antibiotic therapy, 3 demonstrated resistance to 3 or more antimicrobial classes and displayed similar pulsed-field gel electrophoresis patterns. A secondary purpose of this study was to elucidate the evolutionary relationships of strains isolated in this study to genomically characterized Staph. aureus strains. Therefore, Roche 454 GS (Roche Diagnostics Corp., Dallas, TX) pyrosequencing was used to produce draft genome sequences of an MRSA raw milk isolate (H29) and a methicillin-susceptible Staph. aureus (PB32). Analysis using the BLASTn database (http://blast.ncbi.nlm.nih.gov/) demonstrated that the H29 draft genome was highly homologous to the human MRSA strain JH1, yet the β-lactamase plasmid carried by H29 was different from that carried by JH1. Genomic analysis of H29 also clearly explained the multidrug resistance phenotype of this raw milk isolate. Analysis of the PB32 draft genome (using BLASTn) demonstrated that this raw milk isolate was most related to human MRSA strain 04-02981. Although PB32 is not a MRSA, the PB32 draft genome did reveal the presence of a unique staphylococcal cassette mec (SCCmec) remnant. In addition, the PB32 draft genome revealed the presence of a novel bovine staphylococcal pathogenicity island, SaPIbovPB32. This study demonstrates the presence of clones closely related to human and (or) bovine Staph. aureus strains circulating in a dairy herd.