We recently demonstrated that colistin resistance in Acinetobacter baumannii can result from mutational inactivation of genes essential for lipid A biosynthesis (Moffatt JH, et al., Antimicrob. Agents Chemother. 54:4971-4977). Consequently, strains harboring these mutations are unable to produce the major Gram-negative bacterial surface component, lipopolysaccharide (LPS). To understand how A. baumannii compensates for the lack of LPS, we compared the transcriptional profile of the A. baumannii type strain ATCC 19606 to that of an isogenic, LPS-deficient, lpxA mutant strain. The analysis of the expression profiles indicated that the LPS-deficient strain showed increased expression of many genes involved in cell envelope and membrane biogenesis. In particular, upregulated genes included those involved in the Lol lipoprotein transport system and the Mla-retrograde phospholipid transport system. In addition, genes involved in the synthesis and transport of poly-␤-1,6-N-acetylglucosamine (PNAG) also were upregulated, and a corresponding increase in PNAG production was observed. The LPS-deficient strain also exhibited the reduced expression of genes predicted to encode the fimbrial subunit FimA and a type VI secretion system (T6SS). The reduced expression of genes involved in T6SS correlated with the detection of the T6SS-effector protein AssC in culture supernatants of the A. baumannii wild-type strain but not in the LPS-deficient strain. Taken together, these data show that, in response to total LPS loss, A. baumannii alters the expression of critical transport and biosynthesis systems associated with modulating the composition and structure of the bacterial surface.

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We report the resequencing and revised annotation of the Mycobacterium avium subsp. paratuberculosis K10 genome. A total of 90 single-nucleotide errors and a 51-bp indel in the original K10 genome were corrected, and the whole genome annotation was revised. Correction of these sequencing errors resulted in 28 frameshift alterations. The amended genome sequence is accessible via the supplemental section of study SRR060191 in the NCBI Sequence Read Archive and will serve as a valuable reference genome for future studies.The American bovine isolate K10 remains the only Mycobacterium avium subsp. paratuberculosis genome to be fully sequenced and published to date (1). Although this 4.8-Mbp genome likely contains some assembly errors (3), it has provided, and will continue to provide, an invaluable resource for Mycobacterium research. The assembly errors were identified through optical mapping of related M. avium subsp. paratuberculosis strain ATCC 19698, which revealed a 648-kb inversion around the origin of replication and two additional copies of the insertion sequences IS1311 and IS_MAP03 (3). These findings were subsequently validated via PCR, Southern blotting, and (Sanger) sequence analysis in ATCC 19698 and were also confirmed to be present in K10 (3). We designate this interim corrected genome M. avium subsp. paratuberculosis K10Ј. To further improve this resource, we undertook a resequencing project of the original M. avium subsp. paratuberculosis K10 genome.Whole-genome sequencing was performed on the Illumina GAIIx platform using one flow cell lane with 36-cycle pairedend chemistry. Reads were variably trimmed at the 3Ј end based on the Illumina Read Segment Quality Indicator (Illumina manual), and read pairs containing ambiguous bases were removed. Read mapping onto the K10Ј genome sequence was performed using SHRiMP (ver. 1.3.2) (2), and singlenucleotide polymorphisms and indels (deletion and insertion polymorphisms [DIPs]) were called using Nesoni (ver. 0.29; Monash University Victorian Bioinformatics Consortium) with default parameters. Read mapping determined that the data set comprised an average sequence coverage of 72.6 across the K10Ј genome. This high sequence coverage allowed differences between K10{K10Ј and the resequenced version of the genome, designated K10Љ, to be identified with high confidence.Ninety single-nucleotide differences and one 51-bp indel were identified in the K10Љ genome. As confirmation that these differences are likely to represent errors in the original genome sequence, we have also detected these polymorphisms in two additional bovine M. avium subsp. paratuberculosis genomes recently sequenced and assembled within our laboratory (data not shown). Seven of the 90 differences and the 51-bp indel were subjected to PCR and Sanger sequencing for verification. All of the polymorphisms were confirmed to be present in K10Љ compared to the original genome sequence. Thirty-six single-nucleotide deletions and four nucleotide insertions were identified in K10Љ compared to the refere...

Community methicillin-resistant Staphylococcus aureus (cMRSA) is an emerging issue that has resulted in multiple worldwide epidemics. We report the first complete genome sequence of an ST93-MRSA-IV clinical isolate that caused severe invasive infection and a familial outbreak of skin infection. This isolate is a representative of the most common Australian clone of cMRSA that is more distantly related to the previously sequenced genomes of S. aureus.

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Recreational water quality is commonly monitored by means of culture based faecal indicator organism (FIOs) assays. However, these methods are costly and time-consuming; a serious disadvantage when combined with issues such as non-specificity and user bias. New culture and molecular methods have been developed to counter these drawbacks. This study compared industry-standard IDEXX methods (Colilert and Enterolert) with three alternative approaches: 1) TECTA™ system for E. coli and enterococci; 2) US EPA’s 1611 method (qPCR based enterococci enumeration); and 3) Next Generation Sequencing (NGS). Water samples (233) were collected from riverine, estuarine and marine environments over the 2014–2015 summer period and analysed by the four methods. The results demonstrated that E. coli and coliform densities, inferred by the IDEXX system, correlated strongly with the TECTA™ system. The TECTA™ system had further advantages in faster turnaround times (~12 hrs from sample receipt to result compared to 24 hrs); no staff time required for interpretation and less user bias (results are automatically calculated, compared to subjective colorimetric decisions). The US EPA Method 1611 qPCR method also showed significant correlation with the IDEXX enterococci method; but had significant disadvantages such as highly technical analysis and higher operational costs (330% of IDEXX). The NGS method demonstrated statistically significant correlations between IDEXX and the proportions of sequences belonging to FIOs, Enterobacteriaceae, and Enterococcaceae. While costs (3,000% of IDEXX) and analysis time (300% of IDEXX) were found to be significant drawbacks of NGS, rapid technological advances in this field will soon see it widely adopted.

The microbial community and function along with nitrate/nitrite (NOx) removal rates, and nitrogen (N) partitioning into "uptake", "denitrification", and "remaining" via isotope tracers, were studied in soil bioretention mesocolumns (8 unique plant species). Total denitrification gene reads per million (rpm) were positively correlated with % denitrified ( r = 0.69) but negatively correlated with total NOx removal following simulated rain events ( r = -0.79). This is likely due to plant-microbe interactions. Plant species with greater root volume, plant and microbial assimilation %, and NOx removal % had lower denitrification genes and rates. This implies that although microorganisms have access to N, advantageous functions, like denitrification, may not increase. At the conclusion of the 1.5-year experiment, the microbial community was strongly influenced by plant species within the Top zone dominated by plant roots, and the presence or absence of a saturated zone influenced the microbial community within the Bottom zone. Leptospermum continentale was an outlier from the other plants and had much lower denitrification gene rpm (average 228) compared to the other species (range: 277 to 413). The antimicrobial properties and large root volume of Leptospermum continentale likely caused this denitrification gene depression.

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