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Bdellovibrio attack and kill Gram-negative bacteria, including drug-resistant pathogens of animals and plants. This lifestyle is unusual among bacteria, and it imposes unique selective pressures on Bdellovibrio .

After isolating Pantoea sp. strain SOD02 from an urban freshwater stream in Providence, RI, we used PacBio RSII data for de novo assembly and Illumina MiSeq data for polishing. This yielded complete circular sequences for a 4,227,027-bp chromosome with 54.7% GC and a 926,844-bp plasmid with 54.0% GC.

We isolated Aeromonas encheleia strain SOD01 from an urban freshwater stream in Providence, RI. De novo assembly of PacBio RSII data followed by polishing with Illumina MiSeq data generated a complete 4,450,115 bp genome with 61.8% GC content. PGAP annotation predicted 3,877 protein-coding genes, 127 tRNA, and 31 rRNA.

Defining phenotypic and associated genotypic variation among Bdellovibrio may further our understanding of how this genus attacks and kills different Gram-negative bacteria. We isolated Bdellovibrio sp. NC01 from soil. Analysis of 16S rRNA gene sequences and average amino acid identity showed that NC01 belongs to a different species than the type species bacteriovorus. By clustering amino acid sequences from completely sequenced Bdellovibrio and comparing the resulting orthologue groups to a previously published analysis, we defined a 'core genome' of 778 protein-coding genes and identified four protein-coding genes that appeared to be missing only in NC01. To determine how horizontal gene transfer (HGT) may have impacted NC01 genome evolution, we performed genome-wide comparisons of Bdellovibrio nucleotide sequences, which indicated that eight NC01 genomic regions were likely acquired by HGT. To investigate how genome variation may impact predation, we compared protein-coding gene content between NC01 and the B. bacteriovorus type strain HD100, focusing on genes implicated as important in successful killing of prey. Of these, NC01 is missing ten genes that may play roles in lytic activity during predation. Compared to HD100, NC01 kills fewer tested prey strains and kills Escherichia coli ML35 less efficiently. NC01 causes a smaller log reduction in ML35, after which the prey population recovers and the NC01 population decreases. In addition, NC01 forms turbid plaques on lawns of E. coli ML35, in contrast to clear plaques formed by HD100. Linking phenotypic variation in interactions between Bdellovibrio and Gram-negative bacteria with underlying Bdellovibrio genome variation is valuable for understanding the ecological significance of predatory bacteria and evaluating their effectiveness in clinical applications.

We report the complete genome sequence of the predatory bacterium Bdellovibrio sp. strain KM01, isolated from soil collected near a pond. The genome is 3,961,288 bp long with 45.5% GC content. Comparative genomics among Bdellovibrio strains will help us understand how genotypic differences affect differences in predatory phenotypes.

We report the complete genome sequence of the predatory bacterium Bdellovibrio sp. strain KM01, isolated from soil collected near a pond. The genome is 3,961,288 bp long with 45.5% GC content. Comparative genomics among Bdellovibrio strains will help us understand how genotypic differences affect differences in predatory phenotypes.

Defining phenotypic and associated genotypic variation among Bdellovibrio may further our understanding of how this genus attacks and kills different Gram-negative bacteria. We isolated Bdellovibrio sp. NC01 from soil. Analysis of 16S rRNA gene sequences and average amino acid identity showed that NC01 belongs to a different species than the type species bacteriovorus. By clustering amino acid sequences from completely sequenced Bdellovibrio and comparing the resulting orthologue groups to a previously published analysis, we defined a ‘core genome’ of 778 protein-coding genes and identified four protein-coding genes that appeared to be missing only in NC01. To determine how horizontal gene transfer (HGT) may have impacted NC01 genome evolution, we performed genome-wide comparisons of Bdellovibrio nucleotide sequences, which indicated that eight NC01 genomic regions were likely acquired by HGT. To investigate how genome variation may impact predation, we compared protein-coding gene content between NC01 and the B. bacteriovorus type strain HD100, focusing on genes implicated as important in successful killing of prey. Of these, NC01 is missing ten genes that may play roles in lytic activity during predation. Compared to HD100, NC01 kills fewer tested prey strains and kills Escherichia coli ML35 less efficiently. NC01 causes a smaller log reduction in ML35, after which the prey population recovers and the NC01 population decreases. In addition, NC01 forms turbid plaques on lawns of E. coli ML35, in contrast to clear plaques formed by HD100. Linking phenotypic variation in interactions between Bdellovibrio and Gram-negative bacteria with underlying Bdellovibrio genome variation is valuable for understanding the ecological significance of predatory bacteria and evaluating their effectiveness in clinical applications.

The Comprehensive Antibiotic Resistance Database (CARD; http://arpcard.mcmaster.ca) is a manually curated resource containing high quality reference data on the molecular basis of antimicrobial resistance (AMR), with an emphasis on the genes, proteins and mutations involved in AMR. CARD is ontologically structured, model centric, and spans the breadth of AMR drug classes and resistance mechanisms, including intrinsic, mutation-driven and acquired resistance. It is built upon the Antibiotic Resistance Ontology (ARO), a custom built, interconnected and hierarchical controlled vocabulary allowing advanced data sharing and organization. Its design allows the development of novel genome analysis tools, such as the Resistance Gene Identifier (RGI) for resistome prediction from raw genome sequence. Recent improvements include extensive curation of additional reference sequences and mutations, development of a unique Model Ontology and accompanying AMR detection models to power sequence analysis, new visualization tools, and expansion of the RGI for detection of emergent AMR threats. CARD curation is updated monthly based on an interplay of manual literature curation, computational text mining, and genome analysis.

The ability of antimicrobial resistance (AR) to transfer, on mobile genetic elements (MGEs) between bacteria, can cause the rapid establishment of multidrug resistance (MDR) in bacteria from animals, thus creating a foodborne risk to human health. To investigate MDR and its association with plasmids in Salmonella enterica, whole genome sequence (WGS) analysis was performed on 193 S. enterica isolated from sources associated with United States food animals between 1998 and 2011; 119 were resistant to at least one antibiotic tested. Isolates represented 86 serotypes and variants, as well as diverse phenotypic resistance profiles. A total of 923 AR genes and 212 plasmids were identified among the 193 strains. Every isolate contained at least one AR gene. At least one plasmid was detected in 157 isolates. Genes were identified for resistance to aminoglycosides (n = 472), β-lactams (n = 84), tetracyclines (n = 171), sulfonamides (n = 91), phenicols (n = 42), trimethoprim (n = 8), macrolides (n = 5), fosfomycin (n = 48), and rifampicin (n = 2). Plasmid replicon types detected in the isolates were A/C (n = 32), ColE (n = 76), F (n = 43), HI1 (n = 4), HI2 (n = 20), I1 (n = 62), N (n = 4), Q (n = 7), and X (n = 35). Phenotypic resistance correlated with the AR genes identified in 95.4% of cases. Most AR genes were located on plasmids, with many plasmids harboring multiple AR genes. Six antibiotic resistance cassette structures (ARCs) and one pseudo-cassette were identified. ARCs contained between one and five resistance genes (ARC1: sul2, strAB, tetAR; ARC2: aac3-iid; ARC3: aph, sph; ARC4: cmy-2; ARC5: floR; ARC6: tetB; pseudo-ARC: aadA, aac3-VIa, sul1). These ARCs were present in multiple isolates and on plasmids of multiple replicon types. To determine the current distribution and frequency of these ARCs, the public NCBI database was analyzed, including WGS data on isolates collected by the USDA Food Safety and Inspection Service (FSIS) from 2014 to 2018. ARC1, ARC4, and ARC5 were significantly associated with cattle isolates, while ARC6 was significantly associated with chicken isolates. This study revealed that a diverse group of plasmids, carrying AR genes, are responsible for the phenotypic resistance seen in Salmonella isolated from United States food animals. It was also determined that many plasmids carry similar ARCs.

The range of naturally occurring variation in the ability ofBdellovibriostrains to attack and kill Gram-negative bacteria is not well understood. Defining phenotypic and associated genotypic variation amongBdellovibriowill clarify how divergent lineages within this genus impact microbial communities and will inform development of predatory bacteria as biocontrol agents to combat bacterial infections. We isolatedBdellovibriosp. NC01 from soil and compared its genome and predatory phenotypes toB. bacteriovorustype strain HD100. Based on analysis of 16S rRNA gene sequences and average amino acid identity, NC01 belongs to a different species than HD100. Genome-wide comparisons and individual gene analyses indicated that eight NC01 genome regions were likely acquired by horizontal gene transfer (HGT), further supporting an important role for HGT inBdellovibriogenome evolution. Within these regions, multiple protein-coding sequences were assigned predicted functions related to transcriptional regulation and transport; however, most were annotated as hypothetical proteins. Compared to HD100, NC01 has a limited prey range and killsE. coliML35 less efficiently. Whereas HD100 drastically reduces the ML35 population and then maintains low prey population density, NC01 causes a smaller reduction in ML35, after which the prey population recovers, accompanied by a decrease in NC01. In addition, NC01 forms turbid plaques on lawns of ML35, in contrast to clear plaques formed by HD100. Characterizing variation in interactions betweenBdellovibrioand Gram-negative bacteria, such as observed with NC01 and HD100, is important for understanding the ecological significance of predatory bacteria and evaluating their effectiveness in clinical applications.ImportanceBdellovibrioattack and kill Gram-negative bacteria; however, not allBdellovibriostrains are equally efficient at killing the same Gram-negative bacteria. Defining howBdellovibriovary in predatory phenotypes and how this phenotypic variation relates to differences in genotype is important for understanding the ecological significance of predatory bacteria and evaluating their effectiveness in biocontrol of bacterial infections. We determined variation in genome content and predatory phenotypes, including prey range and predation efficiency, betweenBdellovibriosp. NC01 isolated from soil andB. bacteriovorustype strain HD100. NC01 is phylogenetically divergent from HD100, with eight regions of unique gene content likely acquired by horizontal gene transfer. Compared to HD100, the prey range of NC01 is limited, and it was less efficient at killing a strain ofE. coli. These differences may have important implications for how each strain impacts microbial communities in different environments and for the effectiveness of each in clinical applications.

Lack of access to clean water and sanitation is a major factor impacting public health in communities worldwide. To address this, the S-Lab at Providence College and the Global Sustainable Aid Project developed a microflush composting toilet system to isolate and treat human waste. Solid waste is composted within a filter-digester bed via an aerobic process involving microbes and invertebrates. Liquid waste may be sanitized by solar disinfection (SODIS) or slow sand filtration (SSF). Here, we used 16S rRNA amplicon sequencing of samples from a scaled-down test version of the system to better understand the bacterial component of the toilet system. Immediately after fecal matter was deposited in the test system, the bacterial community of the filter-digester bed at the site of deposition resembled that of the human gut at both the phylum and genus level, which was expected. Genus-level analysis of filter-digester bed samples collected over the next 30 days from the site of deposition showed reduced or undetectable levels of fecal-associated taxa, with the exception of Clostridium XI, which persisted at low abundance throughout the sampling period. Starting with the sample collected on day 4, the bacterial community of the filter-digester bed at the site of deposition was dominated by bacterial taxa commonly associated with environmental sources, reflecting a major shift in bacterial community composition. These data support the toilet system’s capacity for processing solid human waste. We also analyzed how SODIS and SSF sanitization methods affected the bacterial community composition of liquid effluent collected on day 15 from the test system. Untreated and treated liquid effluent samples were dominated by Proteobacteria. At the genus level, the bacterial community of the untreated effluent included taxa commonly associated with environmental sources. In the SODIS-treated effluent, these genera increased in abundance, whereas in the SSF-treated effluent, they were greatly reduced or undetectable. By analyzing operational taxonomic units that were unclassified at the genus level, we observed that SSF appears to introduce new taxa into the treated effluent, likely from the biological film of microbes and small animals that constitutes the key element of SSF. These data will inform continued development of liquid waste handling strategies for the toilet system. Using the test system as an indicator of the performance of the full-scale version, we have shown the effectiveness of the microflush composting toilet system for containing and eliminating gut-associated bacteria, thereby improving sanitation and contributing to better public health in rural and peri-urban communities.

The ability of antimicrobial resistance (AR) to transfer, on mobile genetic elements (MGEs) between bacteria, can cause the rapid establishment of multidrug resistance (MDR) in bacteria from animals, thus creating a foodborne risk to human health. To investigate MDR and its association with plasmids in

We report here the complete genome sequence of Pseudomonas sp. strain NC02, isolated from soil in eastern Massachusetts. We assembled PacBio reads into a single closed contig with 132× mean coverage and then polished this contig using Illumina MiSeq reads, yielding a 6,890,566-bp sequence with 61.1% GC content.

We report here the complete genome sequence of Escherichia coli strain ML35. We assembled PacBio reads into a single closed contig with 169× mean coverage and then polished this contig using Illumina MiSeq reads, yielding a 4,918,774-bp sequence with 50.8% GC content.

We report here the complete genome sequence of the facultative predatory bacterium Ensifer adhaerens strain Casida A. The genome was assembled into three circular contigs, with a main chromosome as well as two large secondary replicons, that totaled 7,267,502 bp with 6,641 predicted open reading frames.

We report here the complete genome sequence of the facultative predatory bacterium Ensifer adhaerens strain Casida A. The genome was assembled into three circular contigs, with a main chromosome as well as two large secondary replicons, that totaled 7,267,502 bp with 6,641 predicted open reading frames.

Predatory bacteria attack and digest other bacteria and therefore may play a role in shaping microbial communities. To investigate phenotypic and genotypic variation in saltwater-adapted predatory bacteria, we isolated Halobacteriovorax marinus BE01 from an estuary in Rhode Island, assayed whether it could attack different prey bacteria, and sequenced and analyzed its genome. We found that BE01 is a prey generalist, attacking bacteria from different phylogenetic groups and environments. Gene order and amino acid sequences are highly conserved between BE01 and the H. marinus type strain, SJ. By comparative genomics, we detected two regions of gene content difference that likely occurred via horizontal gene transfer events. Acquired genes encode functions such as modification of DNA, membrane synthesis and regulation of gene expression. Understanding genome evolution and variation in predation phenotypes among predatory bacteria will inform their development as biocontrol agents and clarify how they impact microbial communities., Halobacteriovorax strains are saltwater-adapted predatory bacteria that attack Gram-negative bacteria and may play an important role in shaping microbial communities. To understand how Halobacteriovorax strains impact ecosystems and develop them as biocontrol agents, it is important to characterize variation in predation phenotypes and investigate Halobacteriovorax genome evolution. We isolated Halobacteriovorax marinus BE01 from an estuary in Rhode Island using Vibrio from the same site as prey. Small, fast-moving, attack-phase BE01 cells attach to and invade prey cells, consistent with the intraperiplasmic predation strategy of the H. marinus type strain, SJ. BE01 is a prey generalist, forming plaques on Vibrio strains from the estuary, Pseudomonas from soil, and Escherichia coli. Genome analysis revealed extremely high conservation of gene order and amino acid sequences between BE01 and SJ, suggesting strong selective pressure to maintain the genome in this H. marinus lineage. Despite this, we identified two regions of gene content difference that likely resulted from horizontal gene transfer. Analysis of modal codon usage frequencies supports the hypothesis that these regions were acquired from bacteria with different codon usage biases than H. marinus. In one of these regions, BE01 and SJ carry different genes associated with mobile genetic elements. Acquired functions in BE01 include the dnd operon, which encodes a pathway for DNA modification, and a suite of genes involved in membrane synthesis and regulation of gene expression that was likely acquired from another Halobacteriovorax lineage. This analysis provides further evidence that horizontal gene transfer plays an important role in genome evolution in predatory bacteria. IMPORTANCE Predatory bacteria attack and digest other bacteria and therefore may play a role in shaping microbial communities. To investigate phenotypic and genotypic variation in saltwater-adapted predatory bacteria, we isolated Halobacteriovorax marinus BE01 from an estuary in Rhode Island, assayed whether it could attack different prey bacteria, and sequenced and analyzed its genome. We found that BE01 is a prey generalist, attacking bacteria from different phylogenetic groups and environments. Gene order and amino acid sequences are highly conserved between BE01 and the H. marinus type strain, SJ. By comparative genomics, we detected two regions of gene content difference that likely occurred via horizontal gene transfer events. Acquired genes encode functions such as modification of DNA, membrane synthesis and regulation of gene expression. Understanding genome evolution and variation in predation phenotypes among predatory bacteria will inform their development as biocontrol agents and clarify how they impact microbial communities.

BackgroundHalobacteriovoraxare saltwater-adapted predatory bacteria that attack Gram-negative bacteria and therefore may play an important role in shaping microbial communities. To understand the impact ofHalobacteriovoraxon ecosystems and develop them as biocontrol agents, it is important to characterize variation in predation phenotypes such as prey range and investigate the forces impactingHalobacteriovoraxgenome evolution across different phylogenetic distances.ResultsWe isolatedH. marinusBE01 from an estuary in Rhode Island usingVibriofrom the same site as prey. Small, fast-moving attack phase BE01 cells attach to and invade prey cells, consistent with the intraperiplasmic predation strategy ofH. marinustype strain SJ. BE01 is a prey generalist, forming plaques onVibriostrains from the estuary as well asPseudomonasfrom soil andE. coli. Genome analysis revealed that BE01 is very closely related to SJ, with extremely high conservation of gene order and amino acid sequences. Despite this similarity, we identified two regions of gene content difference that likely resulted from horizontal gene transfer. Analysis of modal codon usage frequencies supports the hypothesis that these regions were acquired from bacteria with different codon usage biases compared toHalobacteriovorax. In BE01, one of these regions includes genes associated with mobile genetic elements, such as a transposase not found in SJ and degraded remnants of an integrase occurring as a full-length gene in SJ. The corresponding region in SJ included unique mobile genetic element genes, such as a site-specific recombinase and bacteriophage-related genes not found in BE01. Acquired functions in BE01 include thedndoperon, which encodes a pathway for DNA modification that may protect DNA from nucleases, and a suite of genes involved in membrane synthesis and regulation of gene expression that was likely acquired from anotherHalobacteriovoraxlineage.ConclusionsOur results support previous observations thatHalobacteriovoraxprey on a broad range of Gram-negative bacteria. Genome analysis suggests strong selective pressure to maintain the genome in theH. marinuslineage represented by BE01 and SJ, although our results also provide further evidence that horizontal gene transfer plays an important role in genome evolution in predatory bacteria.

Indel mutations play key roles in genome and protein evolution, yet we lack a comprehensive understanding of how indels impact evolutionary processes. Genome-wide analyses enabled by next-generation sequencing can clarify the context and effect of indels, thereby integrating a more detailed consideration of indels with our knowledge of nucleotide substitutions. To this end, we sequenced Blochmannia chromaiodes, an obligate bacterial endosymbiont of carpenter ants, and compared it with the close relative, B. pennsylvanicus. The genetic distance between these species is small enough for accurate whole genome alignment but large enough to provide a meaningful spectrum of indel mutations. We found that indels are subjected to purifying selection in coding regions and even intergenic regions, which show a reduced rate of indel base pairs per kilobase compared with nonfunctional pseudogenes. Indels occur almost exclusively in repeat regions composed of homopolymers and multimeric simple sequence repeats, demonstrating the importance of sequence context for indel mutations. Despite purifying selection, some indels occur in protein-coding genes. Most are multiples of three, indicating selective pressure to maintain the reading frame. The deleterious effect of frameshift-inducing indels is minimized by either compensation from a nearby indel to restore reading frame or the indel's location near the 3'-end of the gene. We observed amino acid divergence exceeding nucleotide divergence in regions affected by frameshift-inducing indels, suggesting that these indels may either drive adaptive protein evolution or initiate gene degradation. Our results shed light on how indel mutations impact processes of molecular evolution underlying endosymbiont genome evolution.

Plasmids are important in evolution and adaptation of host bacteria, yet we lack a comprehensive picture of their own natural variation. We used replicon typing and RFLP analysis to assess diversity and distribution of plasmids in the ECOR, SARA, SARB and SARC reference collections of Escherichia coli and Salmonella. Plasmids, especially large (⩾30 kb) plasmids, are abundant in these collections. Host species and genotype clearly impact plasmid prevalence; plasmids are more abundant in ECOR than SAR, but, within ECOR, subgroup B2 strains have the fewest large plasmids. The majority of large plasmids have unique RFLP patterns, suggesting high variation, even within dominant replicon families IncF and IncI1. We found only four conserved plasmid types within ECOR, none of which are widely distributed. Within SAR, conserved plasmid types are primarily serovar-specific, including a pSLT-like plasmid in 13 Typhimurium strains. Conservation of pSLT contrasts with variability of other plasmids, suggesting evolution of serovar-specific virulence plasmids is distinct from that of most enterobacterial plasmids. We sequenced a conserved serovar Heidelberg plasmid but did not detect virulence or antibiotic resistance genes. Our data illustrate the high degree of natural variation in large plasmids of E. coli and Salmonella, even among plasmids sharing backbone genes.

Objectives: To determine the antimicrobial susceptibility of Haemophilus influenzae and Moraxella catarrhalis causing community-acquired respiratory tract infections in the UK and Ireland from 1999/ 2000 to 2006/07. Methods: Sentinel laboratories across the UK and Ireland contributed up to a fixed quota of isolates of defined organisms per annum. A central laboratory confirmed the isolates’ identities, measured MICs by the BSAC agar dilution method and undertook further testing by standard methods. The variability of the MIC method was assessed by repeated annual testing of control isolates. BSAC and EUCAST breakpoints were used. Statistical analysis adjusted for inter-centre variation by random effects logistic regression. Results: A total of 7371 H. influenzae and 2529 M. catarrhalis isolates were investigated. Over 90% of the H. influenzae isolates were susceptible to most of the antimicrobials tested, the exceptions being ampicillin (84.6% susceptible), trimethoprim (84.0%), cefuroxime (82.9%), amoxicillin (77.2%) and cefaclor (11.7%). For M. catarrhalis, resistance was solely due to b-lactamase (prevalence over 91%) reducing susceptibility to penicillins only. There was little evidence of decreased antimicrobial susceptibility between 1999 and 2007 in either pathogen, except for a reduction in susceptibility to trimethoprim in H. influenzae (90.3% to 82.6%, P < 0.00001). On the other hand, tetracycline susceptibility in H. influenzae increased over this period in the UK and Ireland (96.5 to 98.8%, P 5 0.00008). Conclusions: Despite increased resistance in respiratory pathogens from other parts of the world, the susceptibility of H. influenzae and M. catarrhalis to all agents, except tetracycline and trimethoprim in the case of H. influenzae, has remained constant during this longitudinal study.

Stable associations between bacterial endosymbionts and insect hosts provide opportunities to explore genome evolution in the context of established mutualisms and assess the roles of selection and genetic drift across host lineages and habitats. Blochmannia, obligate endosymbionts of ants of the tribe Camponotini, have coevolved with their ant hosts for ∼40 MY. To investigate early events in Blochmannia genome evolution across this ant host tribe, we sequenced Blochmannia from two divergent host lineages, Colobopsis obliquus and Polyrhachis turneri, and compared them with four published genomes from Blochmannia of Camponotus sensu stricto. Reconstructed gene content of the last common ancestor (LCA) of these six Blochmannia genomes is reduced (690 protein coding genes), consistent with rapid gene loss soon after establishment of the symbiosis. Differential gene loss among Blochmannia lineages has affected cellular functions and metabolic pathways, including DNA replication and repair, vitamin biosynthesis and membrane proteins. Blochmannia of P. turneri (i.e., B. turneri) encodes an intact DnaA chromosomal replication initiation protein, demonstrating that loss of dnaA was not essential for establishment of the symbiosis. Based on gene content, B. obliquus and B. turneri are unable to provision hosts with riboflavin. Of the six sequenced Blochmannia, B. obliquus is the earliest diverging lineage (i.e., the sister group of other Blochmannia sampled) and encodes the fewest protein-coding genes and the most pseudogenes. We identified 55 genes involved in parallel gene loss, including glutamine synthetase, which may participate in nitrogen recycling. Pathways for biosynthesis of coenzyme A, terpenoids and riboflavin were lost in multiple lineages, suggesting relaxed selection on the pathway after inactivation of one component. Analysis of Illumina read datasets did not detect evidence of plasmids encoding missing functions, nor the presence of coresident symbionts other than Wolbachia. Although gene order is strictly conserved in four Blochmannia of Camponotus sensu stricto, comparisons with deeply divergent lineages revealed inversions in eight genomic regions, indicating ongoing recombination despite ancestral loss of recA. In sum, the addition of two Blochmannia genomes of divergent host lineages enables reconstruction of early events in evolution of this symbiosis and suggests that Blochmannia lineages may experience distinct, host-associated selective pressures. Understanding how evolutionary forces shape genome reduction in this system may help to clarify forces driving gene loss in other bacteria, including intracellular pathogens.

Sequencing of the large (>50 kb), low-copy-number (Escherichia coli , Staphylococcus , and Corynebacterium cultures was of sufficient quantity and quality for construction of high-coverage libraries, as shown by sequencing five native plasmids ranging in size from 30 kb to 94 kb. We also report recommendations for vector screening to optimize plasmid sequence assembly, preliminary annotation of novel plasmid genomes, and insights on mobile genetic element biology derived from these sequences. Adaptation of this BAC method for large plasmid isolation removes one major technical hurdle to expanding our knowledge of the natural plasmid gene pool.

Shallow facultative rest-phase hypothermia has been reported in a number of passerine families, but few published data exist about its use by free- ranging birds. We used temperature-sensitive radio- transmitters to determine whether White-throated Sparrows (Zonotrichia albicollis) employ nocturnal hypothermia during winter. We measured skin temperatures of 24 free-ranging sparrows between 13:00 and 14:00 and between 02:00 and 03:00 for each of three days and nights per subject. The average nightly skin- temperature reduction per individual was 3.4 ± 1.0°C (SD). Skin temperature reductions ranged from 0.2°C to 7.0°C among all individuals. There was a significant negative correlation between the magnitude of skin temperature decline and nighttime ambient temperature. Additionally, we found a negative trend between depth of hypothermia and a body density index. Fase de Reposo Hipotérmica Facultativa en Individuos de Zonotrichia albicollis que se Desplazan Libremente Resumen. La fase de reposo facultativa somera ha sido mencionada para un número de familias de paseriformes, pero existen pocos datos publicados sobre su uso por parte de aves que se desplazan libremente. Usamos radio transmisores sensibles a la temperatura para determinar si Zonotrichia albicollis emplea hipotermia nocturna durante el invierno. Medimos la temperatura de la piel de 24 individuos que se desplazan libremente entre las 13:00 y 14:00 y entre las 02:00 y 03:00 durante tres días y tres noches por individuo. La reducción nocturna promedio de la temperatura de la piel por individuo fue 3.4 ± 1.0°C (DE). Las reducciones de la temperatura de la piel variaron entre 0.2°C y 7.0°C considerando todos los individuos. Hubo una correlación negativa significativa entre la disminución de la magnitud de la temperatura de la piel y la temperatura ambiental nocturna. Adicionalmente, encontramos una tendencia negativa entre la profundidad de la hipotermia y el índice de densidad corporal.

Objectives To investigate possible molecular mechanisms of azole resistance among fluconazole-susceptible bloodstream isolates of Candida albicans that displayed the trailing growth phenomenon, and to compare these isolates with bloodstream and mucosal isolates that showed reduced susceptibilities to fluconazole. Methods Twelve C. albicans isolates-seven trailing and five susceptible dose dependent (SDD) or resistant (R)-were screened for ERG11 mutations by DNA sequencing and quantification of ERG11, CDR1 and MDR1 expression by RT-PCR using the LightCycler high-speed PCR system. Results SDD and R isolates possessed more homozygous ERG11 mutations than did the trailing isolates. Two of these, V404I and V509M, have not been described previously and were found exclusively in fluconazole SDD and R isolates. Quantification of ERG11 expression revealed that both trailing and SDD and R isolates were capable of ERG11 up-regulation in response to fluconazole, although the SDD and R isolates showed maximal up-regulation at higher fluconazole concentrations. Quantification of CDR1 and MDR1 revealed that all isolates, regardless of in vitro fluconazole response, were capable of CDR1 and MDR1 up-regulation following fluconazole exposure. Furthermore, the SDD and R isolates expressed higher constitutive levels of CDR1 and MDR1 or CDR1, respectively, in the absence of drug compared with trailing isolates. Conclusions Trailing isolates, although susceptible to fluconazole, express the same molecular mechanisms as SDD and R isolates following fluconazole exposure but regulate them differently.

BACKGROUND: The Metropolitan Atlanta Congenital Defects Program (MACDP) is a population-based birth defects surveillance program administered by the Centers for Disease Control and Prevention (CDC) that has been collecting, analyzing, and interpreting birth defects surveillance data since 1967. This paper presents an overview of MACDP current methods and accomplishments over the past 35 years. METHODS: MACDP actively monitors major birth defects among infants born to residents of five counties of metropolitan Atlanta, an area with approximately 50,000 annual births. Cases are ascertained from multiple sources, coded using a modified British Pediatric Association six-digit code, and reviewed and classified by clinical geneticists. RESULTS: MACDP has monitored trends in birth defects rates and has served as a case registry for descriptive, risk factor, and prognostic studies of birth defects, including studies of Agent Orange exposure among Vietnam War veterans, maternal use of multivitamins, diabetes, febrile illnesses, and survival of children with neural tube defects. MACDP has served as a data source for one of the centers participating in the National Birth Defects Prevention Study, and for developing and evaluating neural tube defects prevention strategies related to the periconceptional use of folic acid supplements. CONCLUSIONS: Since its inception, MACDP has served as a resource for the development of uniform methods and approaches to birth defect surveillance across the United States and in many other countries, monitoring birth defects rates, and as a case registry for various descriptive, etiologic, and survival studies of birth defects. MACDP has also served as a training ground for a large number of professionals active in birth defects epidemiology. Birth Defects Research (Part A) 67:617– 624, 2003. Published 2003 Wiley-Liss, Inc. †

ObjectiveThe purpose of this study was to determine whether the candidate genes previously studied in subjects with cleft lip, cleft palate, or both are associated with hypodontia outside the region of the cleft.SubjectsOne hundred twenty subjects from the Iowa Craniofacial Anomalies Research Center were selected based on the availability of both dental records and genotype information.MethodThe type of orofacial clefting and type and location of dental anomalies (missing teeth, supernumerary teeth, or peg laterals) were assessed by dental chart review and radiographic examination. Genotype analysis of candidate genes was performed using polymerase chain reaction/single-strand conformation polymorphism analysis.ResultsThe prevalence of hypodontia in this sample was 47.5%, with 30.0% of subjects having missing teeth outside the cleft. There was a positive association between subjects with cleft lip or cleft lip and palate who had hypodontia outside the cleft region (compared with noncleft controls) and both muscle segment homeo box homolog 1 (MSX1) (p = .029) and transforming growth factor beta 3 (TGFB3) (p = .024). It was not possible in this analysis to determine whether this association was specifically associated with orofacial clefting combined with hypodontia or whether it was due primarily to the clefting phenotype.ConclusionsIn this sample, there was a significantly greater incidence of hypodontia outside the cleft region in subjects with cleft lip and palate, compared with cleft lip only or cleft palate only. Cleft lip and/or palate with hypodontia outside the cleft region was positively associated with both TGFB3 and MSX1, compared with noncleft controls.

Bdellovibrio bacteriovorus invade Gram-negative bacteria in a predatory process requiring Type IV pili (T4P) at a single invasive pole, and also glide on surfaces to locate prey. Ras-like G-protein MglA, working with MglB and RomR in the deltaproteobacterium Myxococcus xanthus, regulates adventurous gliding and T4P-mediated social motility at both M. xanthus cell poles. Our bioinformatic analyses suggested that the GTPase activating protein (GAP)-encoding gene mglB was lost in Bdellovibrio, but critical residues for MglABd GTP-binding are conserved. Deletion of mglABd abolished prey-invasion, but not gliding, and reduced T4P formation. MglABd interacted with a previously uncharacterised tetratricopeptide repeat (TPR) domain protein Bd2492, which we show localises at the single invasive pole and is required for predation. Bd2492 and RomR also interacted with cyclic-di-GMP-binding receptor CdgA, required for rapid prey-invasion. Bd2492, RomRBd and CdgA localize to the invasive pole and may facilitate MglA-docking. Bd2492 was encoded from an operon encoding a TamAB-like secretion system. The TamA protein and RomR were found, by gene deletion tests, to be essential for viability in both predatory and non-predatory modes. Control proteins, which regulate bipolar T4P-mediated social motility in swarming groups of deltaproteobacteria, have adapted in evolution to regulate the anti-social process of unipolar prey-invasion in the “lone-hunter” Bdellovibrio. Thus GTP-binding proteins and cyclic-di-GMP inputs combine at a regulatory hub, turning on prey-invasion and allowing invasion and killing of bacterial pathogens and consequent predatory growth of Bdellovibrio., Author Summary Bacterial cell polarity control is important for maintaining asymmetry of polar components such as flagella and pili. Bdellovibrio bacteriovorus is a predatory deltaproteobacterium which attaches to, and invades, other bacteria using Type IV pili (T4P) extruded from the specialised, invasive, non-flagellar pole of the cell. It was not known how that invasive pole is specified and regulated. Here we discover that a regulatory protein-hub, including Ras-GTPase-like protein MglA and cyclic-di-GMP receptor-protein CdgA, control prey-invasion. In the deltaproteobacterium, Myxococcus xanthus, MglA, with MglB and RomR, was found by others to regulate switching of T4P in social ‘swarming’ surface motility by swapping the pole at which T4P are found. In contrast, in B. bacteriovorus MglA regulates the process of prey-invasion and RomR, which is required for surface motility regulation in Myxococcus, is essential for growth and viability in Bdellovibrio. During evolution, B. bacteriovorus has lost mglB, possibly as T4P-pole-switching is not required; pili are only required at the invasive pole. A previously unidentified tetratricopeptide repeat (TPR) protein interacts with MglA and is essential for prey-invasion. This regulatory protein hub allows prey-invasion, likely integrating cyclic-di-GMP signals, pilus assembly and TamAB secretion in B. bacteriovorus.

Background Evolution equipped Bdellovibrio bacteriovorus predatory bacteria to invade other bacteria, digesting and replicating, sealed within them thus preventing nutrient-sharing with organisms in the surrounding environment. Bdellovibrio were previously described as “obligate predators” because only by mutations, often in gene bd0108, are 1 in ~1x107 of predatory lab strains of Bdellovibrio converted to prey-independent growth. A previous genomic analysis of B. bacteriovorus strain HD100 suggested that predatory consumption of prey DNA by lytic enzymes made Bdellovibrio less likely than other bacteria to acquire DNA by lateral gene transfer (LGT). However the Doolittle and Pan groups predicted, in silico, both ancient and recent lateral gene transfer into the B. bacteriovorus HD100 genome. Results To test these predictions, we isolated a predatory bacterium from the River Tiber- a good potential source of LGT as it is rich in diverse bacteria and organic pollutants- by enrichment culturing with E. coli prey cells. The isolate was identified as B. bacteriovorus and named as strain Tiberius. Unusually, this Tiberius strain showed simultaneous prey-independent growth on organic nutrients and predatory growth on live prey. Despite the prey-independent growth, the homolog of bd0108 did not have typical prey-independent-type mutations. The dual growth mode may reflect the high carbon content of the river, and gives B. bacteriovorus Tiberius extended non-predatory contact with the other bacteria present. The HD100 and Tiberius genomes were extensively syntenic despite their different cultured-terrestrial/freshly-isolated aquatic histories; but there were significant differences in gene content indicative of genomic flux and LGT. Gene content comparisons support previously published in silico predictions for LGT in strain HD100 with substantial conservation of genes predicted to have ancient LGT origins but little conservation of AT-rich genes predicted to be recently acquired. Conclusions The natural niche and dual predatory, and prey-independent growth of the B. bacteriovorus Tiberius strain afforded it extensive non-predatory contact with other marine and freshwater bacteria from which LGT is evident in its genome. Thus despite their arsenal of DNA-lytic enzymes; Bdellovibrio are not always predatory in natural niches and their genomes are shaped by acquiring whole genes from other bacteria.

Background Blochmannia are obligately intracellular bacterial mutualists of ants of the tribe Camponotini. Blochmannia perform key nutritional functions for the host, including synthesis of several essential amino acids. We used Illumina technology to sequence the genome of Blochmannia associated with Camponotus vafer. Results Although Blochmannia vafer retains many nutritional functions, it is missing glutamine synthetase (glnA), a component of the nitrogen recycling pathway encoded by the previously sequenced B. floridanus and B. pennsylvanicus. With the exception of Ureaplasma, B. vafer is the only sequenced bacterium to date that encodes urease but lacks the ability to assimilate ammonia into glutamine or glutamate. Loss of glnA occurred in a deletion hotspot near the putative replication origin. Overall, compared to the likely gene set of their common ancestor, 31 genes are missing or eroded in B. vafer, compared to 28 in B. floridanus and four in B. pennsylvanicus. Three genes (queA, visC and yggS) show convergent loss or erosion, suggesting relaxed selection for their functions. Eight B. vafer genes contain frameshifts in homopolymeric tracts that may be corrected by transcriptional slippage. Two of these encode DNA replication proteins: dnaX, which we infer is also frameshifted in B. floridanus, and dnaG. Conclusions Comparing the B. vafer genome with B. pennsylvanicus and B. floridanus refines the core genes shared within the mutualist group, thereby clarifying functions required across ant host species. This third genome also allows us to track gene loss and erosion in a phylogenetic context to more fully understand processes of genome reduction.

BACKGROUND: Surveillance of stillbirths using fetal death reports (FDRs) has been challenging because of under-reporting of fetal deaths and missing data on the FDRs. Using active case finding and chart abstraction within the infrastructure of established birth defect surveillance programs could potentially enhance the data from FDRs. The data collection form for the Metropolitan Atlanta Congenital Defects Program, an active, population-based birth defects surveillance system, was modified to collect additional information on stillbirths from medical records. METHODS: The study population was a 25% simple random sample of stillbirths recorded on FDRs in 2004 (n = 125) by residents in the five central counties of metropolitan Atlanta. Stillbirth was defined as a fetal death at ≥20 weeks gestation or ≥350 g if age was unknown. Data on demographic characteristics and risk factors collected from the two sources were compared for completeness and agreement, as well as causes of and conditions associated with the fetal death. RESULTS: Combining data sources provided more information. Demographic and risk factor variables in the two data sources showed strong agreement (categorical variable, kappa range = 0.79–1.00; continuous variable, correlation coefficient range = 0.61–1.00). The actively ascertained data provided more complete information for causes and conditions of fetal death. Data from the FDRs yielded 42% of cases with no listed cause of death or associated condition compared with 10% using Metropolitan Atlanta Congenital Defects Program data. CONCLUSIONS: Expanding the potential of existing active birth defects surveillance programs to include stillbirth surveillance could potentially improve the quantity and quality of available data on fetal deaths. Ongoing studies are needed to corroborate these findings and to assess completeness of case ascertainment. Birth Defects Research (Part A), 2008. Published 2008 Wiley-Liss, Inc.

Objectives: The British Society for Antimicrobial Chemotherapy (BSAC) Bacteraemia and RespiratoryResistance Surveillance Programmes are designed for long-term surveillance of antimicrobial resist-ance in key pathogens of bloodstream and community-acquired respiratory infection in the UK andIreland. This paper describes their methods in detail.Methods: Sentinel laboratories across the UK and Ireland contributed up to a fixed quota of isolates ofdefined bacterial groups. Collecting laboratories were compared with national benchmarks for size ofHospital Trust and distribution of bacteraemia pathogens. A central laboratory for each programmeconfirmed the identification of isolates, measured MICs by the BSAC agar dilution method and under-took further testing by standard methods. The variability of the MIC method was assessed by repeatedannual testing of a panel of control isolates. Classification as susceptible, intermediate or resistantwas by BSAC and European Committee on Antimicrobial Susceptibility Testing breakpoints. Statisticalanalysis was adjusted for inter-centre variation using random effects logistic regression.Results: Thirty-two laboratories contributed 16 550 respiratory isolates from 1999–2000 to 2006–07; 30laboratories contributed 15 812 bacteraemia isolates from 2001 to 2006. Although large and teachinghospitals were over-represented, the pattern of bacteraemia organisms seen in the collecting labora-tories in England and Wales was similar to that in national data reported to the Health ProtectionAgency. Replicate MIC measurements showed that 90% agreed within +++++1, and 98% within +++++2,doubling dilutions.Conclusions: These surveillance programmes have provided reliable information on antimicrobialsusceptibility in the UK and Ireland over six and eight seasons, respectively, so far. Detailed resultsshowing non-susceptibility trends, and relationships with potential predictive factors, are presented insix linked papers in this Supplement.Keywords: MIC, breakpoint, agar dilution, statistical analysis, antimicrobial resistance

Pneumococcal disease is prevalent and is a cause of significant morbidity and mortality in the UK and Ireland. We describe the antimicrobial susceptibility and serotype distributions of Streptococcus pneumoniae causing bacteraemia and community-acquired pneumonia in these countries from 1999/2000 to 2006/7, predominantly prior to the introduction of the heptavalent pneumococcal conjugate vaccine (PCV7) into the standard vaccination schedule in September 2006.The BSAC Respiratory and Bacteraemia Resistance Surveillance Programmes collected S. pneumoniae from sentinel laboratories distributed across the UK and Ireland. A central laboratory for each programme re-identified the isolates, determined their serotypes and measured MICs by the BSAC agar dilution method.The prevalence of antimicrobial non-susceptibility, although significant, was generally below the global average. There was no convincing evidence of increasing non-susceptibility over time in either study. The results showed clear differences in the serotype distribution between respiratory and blood isolates, but suggested that PCV7 would provide adequate coverage of invasive isolates in the UK and Ireland. A significant and rapid increase of the non-vaccine serotype 1 among blood isolates from 2001 to 2006 was worrying, given the spread of hypervirulent serotype 1 clones elsewhere in the world.Continued surveillance of both antimicrobial non-susceptibility and serotype distribution changes following the introduction of PCV7 into the routine immunization schedule in the UK and Ireland is imperative. The data presented here, largely obtained prior to the introduction of PCV7 in the UK, provide a valuable baseline against which to monitor changes in antimicrobial non-susceptibility and serotype distribution and hence to identify the expansion of any significant clones.

White matter microstructural integrity was assessed using diffusion tensor imaging (DTI) in 25 young adults, 25 nondemented older adults, and 25 age-matched older adults with dementia of the Alzheimer type (DAT). For each individual, measures of anisotropy and diffusivity were obtained from atlas-transformed images in the anterior and posterior callosum and in the frontal, parietal, temporal and occipital white matter. These data revealed age differences in anisotropy and diffusivity in all assessed regions. Age effects were greater in the anterior as opposed to the posterior corpus callosum and greater in the frontal white matter than in the temporal, parietal and occipital white matter, suggesting age-associated differences in white matter that exhibit a roughly anterior-to-posterior gradient. In contrast, individuals with early-stage dementia exhibited minimal, if any, additional change in anterior regions but did show greater deterioration of white matter in posterior lobar regions. Taken collectively, these results indicate that nondemented aging is characterized by significant changes in white matter most prominently in anterior brain regions. The dissociation between the regional effects of age and dementia status suggests that the mechanisms underlying age-associated cognitive decline are likely distinct from those underlying DAT.

Background Gene rearrangements such as chromosomal translocations have been shown to contribute to cancer development. Human chromosomal fragile sites are regions of the genome especially prone to breakage, and have been implicated in various chromosome abnormalities found in cancer. However, there has been no comprehensive and quantitative examination of the location of fragile sites in relation to all chromosomal aberrations. Results Using up-to-date databases containing all cancer-specific recurrent translocations, we have examined 444 unique pairs of genes involved in these translocations to determine the correlation of translocation breakpoints and fragile sites in the gene pairs. We found that over half (52%) of translocation breakpoints in at least one gene of these gene pairs are mapped to fragile sites. Among these, we examined the DNA sequences within and flanking three randomly selected pairs of translocation-prone genes, and found that they exhibit characteristic features of fragile DNA, with frequent AT-rich flexibility islands and the potential of forming highly stable secondary structures. Conclusion Our study is the first to examine gene pairs involved in all recurrent chromosomal translocations observed in tumor cells, and to correlate the location of more than half of breakpoints to positions of known fragile sites. These results provide strong evidence to support a causative role for fragile sites in the generation of cancer-specific chromosomal rearrangements.