Risk factors for campylobacteriosis in Australia: outcomes of a 2018-2019 case-control study.

BACKGROUND: We aimed to identify risk factors for sporadic campylobacteriosis in Australia, and to compare these for Campylobacter jejuni and Campylobacter coli infections. METHODS: In a multi-jurisdictional case-control study, we recruited culture-confirmed cases of campylobacteriosis reported to state and territory health departments from February 2018 through October 2019. We recruited controls from notified influenza cases in the previous 12 months that were frequency matched to cases by age group, sex, and location. Campylobacter isolates were confirmed to species level by public health laboratories using molecular methods. We conducted backward stepwise multivariable logistic regression to identify significant risk factors. RESULTS: We recruited 571 cases of campylobacteriosis (422 C. jejuni and 84 C. coli) and 586 controls. Important risk factors for campylobacteriosis included eating undercooked chicken (adjusted odds ratio [aOR] 70, 95% CI 13-1296) or cooked chicken (aOR 1.7, 95% CI 1.1-2.8), owning a pet dog aged < 6 months (aOR 6.4, 95% CI 3.4-12), and the regular use of proton-pump inhibitors in the 4 weeks prior to illness (aOR 2.8, 95% CI 1.9-4.3). Risk factors remained similar when analysed specifically for C. jejuni infection. Unique risks for C. coli infection included eating chicken pâté (aOR 6.1, 95% CI 1.5-25) and delicatessen meats (aOR 1.8, 95% CI 1.0-3.3). Eating any chicken carried a high population attributable fraction for campylobacteriosis of 42% (95% CI 13-68), while the attributable fraction for proton-pump inhibitors was 13% (95% CI 8.3-18) and owning a pet dog aged < 6 months was 9.6% (95% CI 6.5-13). The population attributable fractions for these variables were similar when analysed by campylobacter species. Eating delicatessen meats was attributed to 31% (95% CI 0.0-54) of cases for C. coli and eating chicken pâté was attributed to 6.0% (95% CI 0.0-11). CONCLUSIONS: The main risk factor for campylobacteriosis in Australia is consumption of chicken meat. However, contact with young pet dogs may also be an important source of infection. Proton-pump inhibitors are likely to increase vulnerability to infection.

A Prospective, Open-Label Pilot Study of Concurrent Male Partner Treatment for Bacterial Vaginosis.

Up to 50% of women receiving first-line antibiotics for bacterial vaginosis (BV) experience recurrence within 12 weeks. Evidence suggests that reinfection from an untreated regular sexual partner contributes to recurrence. We conducted a pilot study of 34 heterosexual couples to describe the impact of concurrent partner treatment on the composition of the genital microbiota over a 12-week period. We also determined the acceptability and tolerability of concurrent partner treatment and obtained preliminary estimates of the efficacy of the intervention to inform a randomized controlled trial (RCT). Women received first-line antibiotic treatment for BV (i.e., oral metronidazole or intravaginal clindamycin), and their male partner received oral metronidazole, 400 mg, and 2% clindamycin cream applied topically to penile skin, both twice daily for 7 days. The genital microbiota was characterized at three anatomical sites (women, vaginal; men, cutaneous penile and first-pass urine [representing the urethra]) using 16S rRNA gene sequencing. Immediately posttreatment, concurrent partner treatment significantly reduced the abundance of BV-associated bacteria (false-discovery rate [FDR] corrected P value < 0.05) and altered the overall microbiota composition of all three anatomical sites (P = 0.001). Suppression of BV-associated bacteria was sustained in the majority (81%) of women over the 12-week period (FDR P value < 0.05), despite BV-associated bacteria reemerging at both genital sites in men. In this cohort of women at high risk for recurrence, five recurred within 12 weeks of treatment (17%; 95% confidence interval [CI], 6 to 34%). Importantly, men tolerated and adhered to combination therapy. Our findings provide support for an RCT of combined oral and topical male partner treatment for BV. IMPORTANCE Recurrence of BV following standard treatment is unacceptably high. Posttreatment recurrence is distressing for women, and it imposes a considerable burden on the health care system. Recurrences result in multiple presentations to clinical services and repeated antibiotic use, and the associated obstetric and gynecological sequelae are significant. New treatments to improve long-term BV cure are urgently needed. Here, we used 16S rRNA gene sequencing to investigate changes in the microbiota composition at three genital sites (vagina, penile skin, and male urethra) of heterosexual couples undergoing concurrent partner treatment for bacterial vaginosis (BV). We found that concurrent partner treatment immediately and significantly altered the composition of the genital microbiota of both partners, with a reduction in BV-associated bacteria seen at all three sites. BV cure at 12 weeks posttreatment was higher than expected. These microbiological data provide evidence for continued investigation of partner treatment as a strategy to improve BV cure.

Campylobacter jejuni ST50, a pathogen of global importance: A comparative genomic analysis of isolates from Australia, Europe and North America.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis globally, and infections are often transmitted through consumption of raw or undercooked poultry. Campylobacter jejuni ST50 is among the top ten sequence types (STs) reported in the collected isolates listed at PubMLST records from poultry, food and clinical sources for Asia, Europe, North America, Oceania and South America. This study was designed to determine the most commonly reported C. jejuni STs globally using the PubMLST database and assess similarities between genomes of C. jejuni ST50 isolates from geographically distinct locations. To gain a better understanding of C. jejuni diversity, we compared draft genome sequences of 182 ST50 isolates recovered from retail or caecal poultry samples in Oceania, Europe and North America that were collected over a period of 9 years (2010 to 2018). Overall, phylogenetic analysis revealed that isolates from geographically distinct locations tended to cluster based on the continent where the sample was collected. Among ST50 isolates from Europe and North America, we identified resistance determinants associated with phenotypic resistance to beta-lactams (EU: 55%; GB: 43.1%), tetracyclines (CA: 77.3%; EU: 37.5%; GB: 9.8%; US: 43.5%) and fluoroquinolones (EU: 60.0%; GB: 15.7%); no resistance determinants were identified in isolates from Australia. In general, the majority of the virulence genes, with rare exceptions such as wlaN, cj1138, hddA and rfbC, were evenly distributed throughout the genomes of all ST50 isolates in this study. Genomic-based characterization of C. jejuni ST50 isolates from poultry on three continents highlighted that geographically distinct isolates have evolved independently but only represent a glimpse into the diversity of C. jejuni.

Draft Genome Sequences of Eight Campylobacter volucris Isolates from Freshwater Sources in Victoria, Australia.

Campylobacter spp. can survive and be transmitted from a range of environments. Here, we examine eight draft genome sequences of Campylobacter volucris, identified as part of an examination of waterborne Campylobacter species. This is the first report of environmental survival of C. volucris outside gull species.

Molecular characterization of Campylobacter spp. recovered from beef, chicken, lamb and pork products at retail in Australia.

Australian rates of campylobacteriosis are among the highest in developed countries, yet only limited work has been done to characterize Campylobacter spp. in Australian retail products. We performed whole genome sequencing (WGS) on 331 C. coli and 285 C. jejuni from retail chicken meat, as well as beef, chicken, lamb and pork offal (organs). Campylobacter isolates were highly diverse, with 113 sequence types (STs) including 38 novel STs, identified from 616 isolates. Genomic analysis suggests very low levels (2.3-15.3%) of resistance to aminoglycoside, beta-lactam, fluoroquinolone, macrolide and tetracycline antibiotics. A majority (>90%) of isolates (52/56) possessing the fluoroquinolone resistance-associated T86I mutation in the gyrA gene belonged to ST860, ST2083 or ST7323. The 44 pork offal isolates were highly diverse, representing 33 STs (11 novel STs) and harboured genes associated with resistance to aminoglycosides, lincosamides and macrolides not generally found in isolates from other sources. Prevalence of multidrug resistant genotypes was very low (<5%), but ten-fold higher in C. coli than C. jejuni. This study highlights that Campylobacter spp. from retail products in Australia are highly genotypically diverse and important differences in antimicrobial resistance exist between Campylobacter species and animal sources.

Hayes Yard virus: a novel ephemerovirus isolated from a bull with severe clinical signs of bovine ephemeral fever is most closely related to Puchong virus.

Bovine ephemeral fever is a vector-borne disease of ruminants that occurs in tropical and sub-tropical regions of Africa, Asia and Australia. The disease is caused by a rhabdovirus, bovine ephemeral fever virus (BEFV), which occurs as a single serotype globally. Although several other closely related ephemeroviruses have been isolated from cattle and/or arthropods, only kotonkan virus from Nigeria and (tentatively) Mavingoni virus from Mayotte Island in the Indian Ocean have been previously associated with febrile disease. Here, we report the isolation of a novel virus (Hayes Yard virus; HYV) from blood collected in February 2000 from a bull (Bos indicus) in the Northern Territory of Australia. The animal was suffering from a severe ephemeral fever-like illness with neurological involvement, including recumbency and paralysis, and was euthanised. Histological examination of spinal cord and lung tissue identified extensive haemorrhage in the dura mata with moderate perineuronal oedema and extensive emphysema. HYV displayed cone-shaped morphology, typical of rhabdoviruses, and was found to be most closely related antigenically to Puchong virus (PUCV), isolated in 1965 from mosquitoes in Malaysia. Analysis of complete genome sequences of HYV (15 025 nt) and PUCV (14 932 nt) indicated that each has a complex organisation (3' N-P-M-G-GNS-α1-α2-ß-γ-L 5') and expression strategy, similar to that of BEFV. Based on an alignment of complete L protein sequences, HYV and PUCV cluster with other rhabdoviruses in the genus Ephemerovirus and appear to represent two new species. Neutralising antibody to HYV was also detected in a retrospective survey of cattle sera collected in the Northern Territory.

Extended sequencing of vaccine and wild-type capripoxvirus isolates provides insights into genes modulating virulence and host range.

The genus Capripoxvirus in the subfamily Chordopoxvirinae, family Poxviridae, comprises sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which cause the eponymous diseases across parts of Africa, the Middle East and Asia. These diseases cause significant economic losses and can have a devastating impact on the livelihoods and food security of small farm holders. So far, only live classically attenuated SPPV, GTPV and LSDV vaccines are commercially available and the history, safety and efficacy of many have not been well established. Here, we report 13 new capripoxvirus genome sequences, including the hairpin telomeres, from both pathogenic field isolates and vaccine strains. We have also updated the genome annotations to incorporate recent advances in our understanding of poxvirus biology. These new genomes and genes grouped phenetically with other previously sequenced capripoxvirus strains, and these new alignments collectively identified several recurring alterations in genes thought to modulate virulence and host range. In particular, some of the many large capripoxvirus ankyrin and kelch-like proteins are commonly mutated in vaccine strains, while the variola virus B22R-like gene homolog has also been disrupted in many vaccine isolates. Among these vaccine isolates, frameshift mutations are especially common and clearly present a risk of reversion to wild type in vaccines bearing these mutations. A consistent pattern of gene inactivation from LSDV to GTPV and then SPPV is also observed, much like the pattern of gene loss in orthopoxviruses, but, rather surprisingly, the overall genome size of ~150 kbp remains relatively constant. These data provide new insights into the evolution of capripoxviruses and the determinants of pathogenicity and host range. They will find application in the development of new vaccines with better safety, efficacy and trade profiles.

Salmonella from a Microtidal Estuary Are Capable of Invading Human Intestinal Cell Lines.

Faecal contamination poses health risks for the recreational users of urban estuaries. However, our understanding of the potential pathogenicity of faecal microbes in these environments is limited. To this end, a study was conducted to understand the spatial and seasonal distribution of Salmonella in water and sediments of the Yarra River estuary, Melbourne, Australia. Among 210 samples in total, culturable Salmonella were recovered from 27%, 17%, and 19% of water, bank, and bed sediment samples, respectively. The combined detection increased from 15% in winter to 32% in summer (p < 0.05) indicating seasonal variation as potential part of public health risk assessments. Further, pathogenic potential of the Salmonella isolates was characterised via the quantification of attachment and invasion capacity using human epithelial colorectal cell line Caco-2 on a subset of isolates (n = 62). While all of these isolates could attach and invade Caco-2 cells, 52% and 13% of these showed greater attachment and invasiveness, respectively, than the corresponding mean values for S. Typhimurium ATCC14028 control. Isolates from winter were on average more invasive (seven out of eight isolates with the highest invasiveness recovered from the colder sampling period) than the isolates from summer, and Salmonella collected during summer showed lower invasion (p < 0.05) compared with the control. Similar low invasion compared with the same control was observed for isolates recovered from bank sediment (p < 0.05). While the higher prevalence in summer may imply higher risks during these peak recreational periods, it is essential that this information is used in combination with quantitative microbial risk assessments to fully understand the health risks posed by Salmonella in microtidal estuaries.

Gardnerella vaginalis Clade Distribution Is Associated With Behavioral Practices and Nugent Score in Women Who Have Sex With Women.

BACKGROUND: Gardnerella vaginalis is detected in women with and without bacterial vaginosis (BV). Identification of 4 G. vaginalis clades raised the possibility that pathogenic and commensal clades exist. We investigated the association of behavioral practices and Nugent Score with G. vaginalis clade distribution in women who have sex with women (WSW). METHODS: Longitudinal self-collected vaginal specimens were analyzed using established G. vaginalis species-specific and clade-typing polymerase chain reaction assays. Logistic regression assessed factors associated with detection of G. vaginalis clades, and multinomial regression assessed factors associated with number of clades. RESULTS: Clades 1, 2, and 3 and multiclade communities (<2 clades) were associated with Nugent-BV. Clade 1 (odds ratio [OR], 3.36; 95% confidence interval [CI], 1.65-6.84) and multiclade communities (relative risk ratio [RRR], 9.51; 95% CI, 4.36-20.73) were also associated with Lactobacillus-deficient vaginal microbiota. Clade 4 was neither associated with Nugent-BV nor Lactobacillus-deficient microbiota (OR, 1.49; 95% CI, 0.67-3.33). Specific clades were associated with differing behavioral practices. Clade 1 was associated with increasing number of recent sexual partners and smoking, whereas clade 2 was associated with penile-vaginal sex and sharing of sex toys with female partners. CONCLUSIONS: Our results suggest that G. vaginalis clades have varying levels of pathogenicity in WSW, with acquisition occurring through sexual activity. These findings suggest that partner treatment may be an appropriate strategy to improve BV cure.

Sexual practices have a significant impact on the vaginal microbiota of women who have sex with women.

Women-who-have-sex-with-women (WSW) are at increased risk of bacterial vaginosis (BV). We investigated the impact of practices and past BV on the vaginal microbiota within a two-year longitudinal cohort of Australian WSW. Self-collected vaginal swabs were used to characterise the vaginal microbiota using 16S-rRNA gene sequencing. Hierarchical clustering defined community state types (CSTs). Bacterial diversity was calculated using the Shannon diversity index and instability of the vaginal microbiota was assessed by change of CST and Bray-Curtis dissimilarity. Sex with a new partner increased the bacterial diversity (adjusted-coefficient = 0.41, 95%CI: 0.21,0.60, p < 0.001) and instability of the vaginal microbiota, in terms of both change of CST (adjusted-odds-ratio = 2.65, 95%CI: 1.34,5.22, p = 0.005) and increased Bray-Curtis dissimilarity (adjusted-coefficient = 0.21, 95%CI: 0.11,0.31, p < 0.001). Women reporting sex with a new partner were more likely than women reporting no new partner to have a vaginal microbiota characterised by Gardnerella vaginalis (adjusted-relative-risk-ratio[aRRR] = 3.45, 95%CI: 1.42,8.41, p = 0.006) or anaerobic BV-associated bacteria (aRRR = 3.62, 95%CI: 1.43,9.14, p = 0.007) relative to a Lactobacillus crispatus dominated microbiota. Sex with a new partner altered the vaginal microbiota of WSW by increasing the diversity and abundance of BV-associated bacteria. These findings highlight the influence of practices on the development of a non-optimal vaginal microbiota and provide microbiological support for the sexual exchange of bacteria between women.

Gut microbiota of preterm infants supplemented with probiotics: sub-study of the ProPrems trial.

BACKGROUND: The ProPrems trial, a multi-center, double-blind, placebo-controlled randomized trial, previously reported a 54% reduction in necrotizing enterocolitis (NEC) of Bell stage 2 or more from 4.4 to 2.0% in 1099 infants born before 32 completed weeks' gestation and weighing < 1500 g, receiving probiotic supplementation (with Bifidobacterium longum subsp. infantis BB-02, Streptococcus thermophilus TH-4 and Bifidobacterium animalis subsp. lactis BB-12). This sub-study investigated the effect of probiotic supplementation on the gut microbiota in a cohort of very preterm infants in ProPrems. RESULTS: Bifidobacterium was found in higher abundance in infants who received the probiotics (AOR 17.22; 95% CI, 3.49-84.99, p < 0.001) as compared to the placebo group, and Enterococcus was reduced in infants receiving the probiotic during the supplementation period (AOR 0.27; 95% CI, 0.09-0.82, p = 0.02). CONCLUSION: Probiotic supplementation with BB-02, TH-4 and BB-12 from soon after birth increased the abundance of Bifidobacterium in the gut microbiota of very preterm infants. Increased abundance of Bifidobacterium soon after birth may be associated with reducing the risk of NEC in very preterm infants.

The changing landscape of vancomycin-resistant Enterococcus faecium in Australia: a population-level genomic study.

Background: Vancomycin-resistant Enterococcus faecium (VREfm) represent a major source of nosocomial infection worldwide. In Australia, there has been a recent concerning increase in bacteraemia associated with the vanA genotype, prompting investigation into the genomic epidemiology of VREfm. Methods: A population-level study of VREfm (10 November-9 December 2015) was conducted. A total of 321 VREfm isolates (from 286 patients) across Victoria State were collected and sequenced with Illumina NextSeq. SNPs were used to assess relatedness. STs and genes associated with resistance and virulence were identified. The vanA-harbouring plasmid from an isolate from each ST was assembled using long-read data. Illumina reads from remaining isolates were then mapped to these assemblies to identify their probable vanA-harbouring plasmid. Results: vanA-VREfm comprised 17.8% of isolates. ST203, ST80 and a pstS(-) clade, ST1421, predominated (30.5%, 30.5% and 37.2%, respectively). Most vanB-VREfm were ST796 (77.7%). vanA-VREfm were more closely related within hospitals versus between them [core SNPs 10 (IQR 1-357) versus 356 (179-416), respectively], suggesting discrete introductions of vanA-VREfm, with subsequent intra-hospital transmission. In contrast, vanB-VREfm had similar core SNP distributions within versus between hospitals, due to widespread dissemination of ST796. Different vanA-harbouring plasmids were found across STs. With the exception of ST78 and ST796, Tn1546 transposons also varied. Phylogenetic analysis revealed Australian strains were often interspersed with those from other countries, suggesting ongoing cross-continental transmission. Conclusions: Emerging vanA-VREfm in Australia is polyclonal, indicating repeat introductions of vanA-VREfm into hospitals and subsequent dissemination. The close relationship to global strains reinforces the need for ongoing screening and control of VREfm in Australia and abroad.

Genomic epidemiology and antimicrobial resistance of Neisseria gonorrhoeae in New Zealand.

Background: Antimicrobial-resistant Neisseria gonorrhoeae is a major threat to public health. No studies to date have examined the genomic epidemiology of gonorrhoea in the Western Pacific Region, where the incidence of gonorrhoea is particularly high. Methods: A population-level study of N. gonorrhoeae in New Zealand (October 2014 to May 2015). Comprehensive susceptibility testing and WGS data were obtained for 398 isolates. Relatedness was inferred using phylogenetic trees, and pairwise core SNPs. Mutations and genes known to be associated with resistance were identified, and correlated with phenotype. Results: Eleven clusters were identified. In six of these clusters, >25% of isolates were from females, while in eight of them, >15% of isolates were from females. Drug resistance was common; 98%, 32% and 68% of isolates were non-susceptible to penicillin, ciprofloxacin and tetracycline, respectively. Elevated MICs to extended-spectrum cephalosporins (ESCs) were observed in 3.5% of isolates (cefixime MICs ≥ 0.12 mg/L, ceftriaxone MICs ≥ 0.06 mg/L). Only nine isolates had penA XXXIV genotypes, three of which had decreased susceptibility to ESCs (MIC = 0.12 mg/L). Azithromycin non-susceptibility was identified in 43 isolates (10.8%); two of these isolates had 23S mutations (C2611T, 4/4 alleles), while all had mutations in mtrR or its promoter. Conclusions: The high proportion of females in clusters suggests transmission is not exclusively among MSM in New Zealand; re-assessment of risk factors for transmission may be warranted in this context. As elevated MICs of ESCs and/or azithromycin were found in closely related strains, targeted public health interventions to halt transmission are urgently needed.

Draft Genome Sequences of Two Strains of a Newly Described Species, Sphingobacterium cellulitidis.

The draft genome sequences of two strains of a newly described species, Sphingobacterium cellulitidis, have been determined. The type strain originated from cellulitis of a toe of a patient and the other strain from the environment. The sequences will provide the reference genomes of the new Sphingobacterium species.

Global Gene Expression Profile of Acinetobacter baumannii During Bacteremia.

Background: Acinetobacter baumannii is a pathogen of major importance in intensive care units worldwide, with the potential to cause problematic outbreaks and acquire high-level resistance to antibiotics. There is an urgent need to understand the mechanisms of A. baumannii pathogenesis for the future development of novel targeted therapies. In this study we performed an in vivo transcriptomic analysis of A. baumannii isolated from a mammalian host with bacteremia. Methods: Mice were infected with A. baumannii American Type Culture Collection 17978 using an intraperitoneal injection, and blood was extracted at 8 hours to purify bacterial RNA for RNA-Seq with an Illumina platform. Results: Approximately one-quarter of A. baumannii protein coding genes were differentially expressed in vivo compared with in vitro (false discovery rate, ≤0.001; 2-fold change) with 557 showing decreased and 329 showing increased expression. Gene groups with functions relating to translation and RNA processing were overrepresented in genes with increased expression, and those relating to chaperone and protein turnover were overrepresented in the genes with decreased expression. The most strongly up-regulated genes corresponded to the 3 recognized siderophore iron uptake clusters, reflecting the iron-restrictive environment in vivo. Metabolic changes in vivo included reduced expression of genes involved in amino acid and fatty acid transport and catabolism, indicating metabolic adaptation to a different nutritional environment. Genes encoding types I and IV pili, quorum sensing components, and proteins involved in biofilm formation all showed reduced expression. Many genes that have been reported as essential for virulence showed reduced or unchanged expression in vivo. Conclusion: This study provides the first insight into A. baumannii gene expression profiles during a life-threatening mammalian infection. Analysis of differentially regulated genes highlights numerous potential targets for the design of novel therapeutics.

Porphyromonas gingivalis Uses Specific Domain Rearrangements and Allelic Exchange to Generate Diversity in Surface Virulence Factors.

Porphyromonas gingivalis is a keystone pathogen of chronic periodontitis. The virulence of P. gingivalis is reported to be strain related and there are currently a number of strain typing schemes based on variation in capsular polysaccharide, the major and minor fimbriae and adhesin domains of Lys-gingipain (Kgp), amongst other surface proteins. P. gingivalis can exchange chromosomal DNA between strains by natural competence and conjugation. The aim of this study was to determine the genetic variability of P. gingivalis strains sourced from international locations over a 25-year period and to determine if variability in surface virulence factors has a phylogenetic basis. Whole genome sequencing was performed on 13 strains and comparison made to 10 previously sequenced strains. A single nucleotide polymorphism-based phylogenetic analysis demonstrated a shallow tri-lobed phylogeny. There was a high level of reticulation in the phylogenetic network, demonstrating extensive horizontal gene transfer between the strains. Two highly conserved variants of the catalytic domain of the major virulence factor the Kgp proteinase (KgpcatI and KgpcatII) were found. There were three variants of the fourth Kgp C-terminal cleaved adhesin domain. Specific variants of the cell surface proteins FimA, FimCDE, MfaI, RagAB, Tpr, and PrtT were also identified. The occurrence of all these variants in the P. gingivalis strains formed a mosaic that was not related to the SNP-based phylogeny. In conclusion P. gingivalis uses domain rearrangements and genetic exchange to generate diversity in specific surface virulence factors.

CYP101J2, CYP101J3, and CYP101J4, 1,8-Cineole-Hydroxylating Cytochrome P450 Monooxygenases from Sphingobium yanoikuyae Strain B2.

We report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived from Sphingobium yanoikuyae B2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8-cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His6 tagged) in Escherichia coli BL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation in E. coli demonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners from E. coli to yield a product putatively identified as (1S)-2α-hydroxy-1,8-cineole or (1R)-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found in Novosphingobium aromaticivorans and Pseudomonas putida Compared to P450cin (CYP176A1), a 1,8-cineole-hydroxylating P450 from Citrobacter braakii, these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications. IMPORTANCE: CYP101J2, CYP101J3, and CYP101J4 are cytochrome P450 monooxygenases from S. yanoikuyae B2 that hydroxylate the monoterpenoid 1,8-cineole. These enzymes not only play an important role in microbial degradation of this plant-based chemical but also provide an interesting route to synthesize oxygenated 1,8-cineole derivatives for applications as natural flavor and fragrance precursors or incorporation into polymers. The P450 cytochromes also provide an interesting basis from which to compare other enzymes with a similar function and expand the CYP101 family. This could eventually provide enough bacterial parental enzymes with similar amino acid sequences to enable in vitro evolution via DNA shuffling.

The NEAT Domain-Containing Proteins of Clostridium perfringens Bind Heme.

The ability of a pathogenic bacterium to scavenge iron from its host is important for its growth and survival during an infection. Our studies on C. perfringens gas gangrene strain JIR325, a derivative of strain 13, showed that it is capable of utilizing both human hemoglobin and ferric chloride, but not human holo-transferrin, as an iron source for in vitro growth. Analysis of the C. perfringens strain 13 genome sequence identified a putative heme acquisition system encoded by an iron-regulated surface gene region that we have named the Cht (Clostridium perfringens heme transport) locus. This locus comprises eight genes that are co-transcribed and includes genes that encode NEAT domain-containing proteins (ChtD and ChtE) and a putative sortase (Srt). The ChtD, ChtE and Srt proteins were shown to be expressed in JIR325 cells grown under iron-limited conditions and were localized to the cell envelope. Moreover, the NEAT proteins, ChtD and ChtE, were found to bind heme. Both chtDE and srt mutants were constructed, but these mutants were not defective in hemoglobin or ferric chloride utilization. They were, however, attenuated for virulence when tested in a mouse myonecrosis model, although the virulence phenotype could not be restored via complementation and, as is common with such systems, secondary mutations were identified in these strains. In summary, this study provides evidence for the functional redundancies that occur in the heme transport pathways of this life threatening pathogen.

Prospective Whole-Genome Sequencing Enhances National Surveillance of Listeria monocytogenes.

Whole-genome sequencing (WGS) has emerged as a powerful tool for comparing bacterial isolates in outbreak detection and investigation. Here we demonstrate that WGS performed prospectively for national epidemiologic surveillance of Listeria monocytogenes has the capacity to be superior to our current approaches using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), multilocus variable-number tandem-repeat analysis (MLVA), binary typing, and serotyping. Initially 423 L. monocytogenes isolates underwent WGS, and comparisons uncovered a diverse genetic population structure derived from three distinct lineages. MLST, binary typing, and serotyping results inferred in silico from the WGS data were highly concordant (>99%) with laboratory typing performed in parallel. However, WGS was able to identify distinct nested clusters within groups of isolates that were otherwise indistinguishable using our current typing methods. Routine WGS was then used for prospective epidemiologic surveillance on a further 97 L. monocytogenes isolates over a 12-month period, which provided a greater level of discrimination than that of conventional typing for inferring linkage to point source outbreaks. A risk-based alert system based on WGS similarity was used to inform epidemiologists required to act on the data. Our experience shows that WGS can be adopted for prospective L. monocytogenes surveillance and investigated for other pathogens relevant to public health.