Epidemiology of healthcare-associated Pseudomonas aeruginosa in intensive care units: are sink drains to blame?

BACKGROUND: Pseudomonas aeruginosa (PA) is a common cause of healthcare-associated infection (PA-HAI) in the intensive care unit (ICU). AIM: To describe the epidemiology of PA-HAI in ICUs in Ontario, Canada, and to identify episodes of sink-to-patient PA transmission. METHODS: This was a prospective cohort study of patients in six ICUs from 2018 to 2019, with retrieval of PA clinical isolates, and PA-screening of antimicrobial-resistant organism surveillance rectal swabs, and of sink drain, air, and faucet samples. All PA isolates underwent whole-genome sequencing. PA-HAI was defined using US National Healthcare Safety Network criteria. ICU-acquired PA was defined as PA isolated from specimens obtained ≥48 h after ICU admission in those with prior negative rectal swabs. Sink-to-patient PA transmission was defined as ICU-acquired PA with close genomic relationship to isolate(s) previously recovered from sinks in a room/bedspace occupied 3-14 days prior to collection date of the relevant patient specimen. FINDINGS: Over ten months, 72 PA-HAIs occurred among 60/4263 admissions. The rate of PA-HAI was 2.40 per 1000 patient-ICU-days; higher in patients who were PA-colonized on admission. PA-HAI was associated with longer stay (median: 26 vs 3 days uninfected; P < 0.001) and contributed to death in 22/60 cases (36.7%). Fifty-eight admissions with ICU-acquired PA were identified, contributing 35/72 (48.6%) PA-HAIs. Four patients with five PA-HAIs (6.9%) had closely related isolates previously recovered from their room/bedspace sinks. CONCLUSION: Nearly half of PA causing HAI appeared to be acquired in ICUs, and 7% of PA-HAIs were associated with sink-to-patient transmission. Sinks may be an under-recognized reservoir for HAIs.

Staphylococcus aureus interaction with Pseudomonas aeruginosa biofilm enhances tobramycin resistance.

Antimicrobial resistance is a significant threat to the treatment of infectious disease. Multiple mechanisms of resistance to different classes of antibiotics have been identified and well-studied. However, these mechanisms are studied with bacteria in isolation, whereas often, infections have a polymicrobial basis. Using a biofilm slide chamber model, we visualized the formation and development of clinical Pseudomonas aeruginosa biofilms in the presence of secreted Staphylococcus aureus exoproducts, two bacteria that commonly co-infect pediatric patients with cystic fibrosis. We showed that, over time, certain isolates of P. aeruginosa can form different biofilm architecture in the presence of S. aureus exoproducts. We further determined that this interaction was dependent on Psl produced by P. aeruginosa and staphylococcal protein A from S. aureus. Importantly, we identified a mechanism of antibiotic resistance to tobramycin that is dependent on the polymicrobial interactions between these two bacteria. This interaction occurred in isolates of P. aeruginosa recovered from children with cystic fibrosis who failed to clear P. aeruginosa following inhaled tobramycin treatment.

Impact of Immunosuppression on the Metagenomic Composition of the Intestinal Microbiome: a Systems Biology Approach to Post-Transplant Diabetes.

Solid organ transplantation (SOT) outcomes have continued to improve, although long-term use of immunosuppressants can lead to complications such as diabetes, compromising post-transplant outcomes. In this study, we have characterized the intestinal microbiome (IM) composition at the metagenomic level in the context of hyperglycemia induced by immunosuppressants. Sprague-Dawley rats were subjected to doses of tacrolimus and sirolimus that reliably induce hyperglycemia and an insulin-resistant state. Subsequent exposure to probiotics resulted in reversal of hyperglycemia. 16S rRNA and metagenomic sequencing of stool were done to identify the bacterial genes and pathways enriched in immunosuppression. Bacterial diversity was significantly decreased in sirolimus-treated rats, with 9 taxa significantly less present in both immunosuppression groups: Roseburia, Oscillospira, Mollicutes, Rothia, Micrococcaceae, Actinomycetales and Staphylococcus. Following probiotics, these changes were reversed to baseline. At the metagenomic level, the balance of metabolism was shifted towards the catabolic side with an increase of genes involved in sucrose degradation, similar to diabetes. Conversely, the control rats had greater abundance of anabolic processes and genes involved in starch degradation. Immunosuppression leads to a more catabolic microbial profile, which may influence development of diabetes after SOT. Modulation of the microbiome with probiotics may help in minimizing adverse long-term effects of immunosuppression.

Chronic infection phenotypes of Pseudomonas aeruginosa are associated with failure of eradication in children with cystic fibrosis.

Early eradication treatment with inhaled tobramycin is successful in the majority of children with cystic fibrosis (CF) with incident Pseudomonas aeruginosa infection. However, in 10-40 % of cases, eradication fails and the reasons for this are poorly understood. The purpose of this study was to determine whether specific microbial characteristics could explain eradication treatment failure. This was a cross-sectional study of CF patients (aged 0-18 years) with incident P. aeruginosa infection from 2011 to 2014 at the Hospital for Sick Children, Toronto, Canada. Phenotypic assays were done on all incident P. aeruginosa isolates, and eradicated and persistent isolates were compared using the Mann-Whitney test or the two-sided Chi-square test. A total of 46 children with CF had 51 incident P. aeruginosa infections. In 72 % (33/46) of the patients, eradication treatment was successful, while 28 % failed eradication therapy. Persistent isolates were less likely to be motile, with significantly less twitch motility (p=0.001), were more likely to be mucoid (p=0.002), and more likely to have a tobramycin minimum inhibitory concentration (MIC) ≥ 128 µg/mL (p=0.02) compared to eradicated isolates. Although biofilm production was similar, there was a trend towards more persistent isolates with deletions in quorum-sensing genes compared with eradicated isolates (p=0.06). Initial acquisition of P. aeruginosa with characteristics of chronic infection is associated with failure of eradication treatment.

Impact of maternal intrapartum antibiotics, method of birth and breastfeeding on gut microbiota during the first year of life: a prospective cohort study.

OBJECTIVE: Dysbiosis of the infant gut microbiota may have long-term health consequences. This study aimed to determine the impact of maternal intrapartum antibiotic prophylaxis (IAP) on infant gut microbiota, and to explore whether breastfeeding modifies these effects. DESIGN: Prospective pregnancy cohort of Canadian infants born in 2010-2012: the Canadian Healthy Infant Longitudinal Development (CHILD) Study. SETTING: General community. SAMPLE: Representative sub-sample of 198 healthy term infants from the CHILD Study. METHODS: Maternal IAP exposures and birth method were documented from hospital records and breastfeeding was reported by mothers. Infant gut microbiota was characterised by Illumina 16S rRNA sequencing of faecal samples at 3 and 12 months. MAIN OUTCOME MEASURES: Infant gut microbiota profiles. RESULTS: In this cohort, 21% of mothers received IAP for Group B Streptococcus prophylaxis or pre-labour rupture of membranes; another 23% received IAP for elective or emergency caesarean section (CS). Infant gut microbiota community structures at 3 months differed significantly with all IAP exposures, and differences persisted to 12 months for infants delivered by emergency CS. Taxon-specific composition also differed, with the genera Bacteroides and Parabacteroides under-represented, and Enterococcus and Clostridium over-represented at 3 months following maternal IAP. Microbiota differences were especially evident following IAP with emergency CS, with some changes (increased Clostridiales and decreased Bacteroidaceae) persisting to 12 months, particularly among non-breastfed infants. CONCLUSIONS: Intrapartum antibiotics in caesarean and vaginal delivery are associated with infant gut microbiota dysbiosis, and breastfeeding modifies some of these effects. Further research is warranted to explore the health consequences of these associations. TWEETABLE ABSTRACT: Maternal #antibiotics during childbirth alter the infant gut #microbiome.

Infant gut microbiota and food sensitization: associations in the first year of life.

BACKGROUND: The gut microbiota is established during infancy and plays a fundamental role in shaping host immunity. Colonization patterns may influence the development of atopic disease, but existing evidence is limited and conflicting. OBJECTIVE: To explore associations of infant gut microbiota and food sensitization. METHODS: Food sensitization at 1 year was determined by skin prick testing in 166 infants from the population-based Canadian Healthy Infant Longitudinal Development (CHILD) study. Faecal samples were collected at 3 and 12 months, and microbiota was characterized by Illumina 16S rRNA sequencing. RESULTS: Twelve infants (7.2%) were sensitized to ≥ 1 common food allergen at 1 year. Enterobacteriaceae were overrepresented and Bacteroidaceae were underrepresented in the gut microbiota of food-sensitized infants at 3 months and 1 year, whereas lower microbiota richness was evident only at 3 months. Each quartile increase in richness at 3 months was associated with a 55% reduction in risk for food sensitization by 1 year (adjusted odds ratio 0.45, 95% confidence interval 0.23-0.87). Independently, each quartile increase in Enterobacteriaceae/Bacteroidaceae ratio was associated with a twofold increase in risk (2.02, 1.07-3.80). These associations were upheld in a sensitivity analysis among infants who were vaginally delivered, exclusively breastfed and unexposed to antibiotics. At 1 year, the Enterobacteriaceae/Bacteroidaceae ratio remained elevated among sensitized infants, who also tended to have decreased abundance of Ruminococcaceae. CONCLUSIONS AND CLINICAL RELEVANCE: Low gut microbiota richness and an elevated Enterobacteriaceae/Bacteroidaceae ratio in early infancy are associated with subsequent food sensitization, suggesting that early gut colonization may contribute to the development of atopic disease, including food allergy.

Associations between bacterial communities of house dust and infant gut.

The human gut is host to a diverse and abundant community of bacteria that influence health and disease susceptibility. This community develops in infancy, and its composition is strongly influenced by environmental factors, notably perinatal anthropogenic exposures such as delivery mode (Cesarean vs. vaginal) and feeding method (breast vs. formula); however, the built environment as a possible source of exposure has not been considered. Here we report on a preliminary investigation of the associations between bacteria in house dust and the nascent fecal microbiota from 20 subjects from the Canadian Healthy Infant Longitudinal Development (CHILD) Study using high-throughput sequence analysis of portions of the 16S rRNA gene. Despite significant differences between the dust and fecal microbiota revealed by Nonmetric Multidimensional Scaling (NMDS) analysis, permutation analysis confirmed that 14 bacterial OTUs representing the classes Actinobacteria (3), Bacilli (3), Clostridia (6) and Gammaproteobacteria (2) co-occurred at a significantly higher frequency in matched dust-stool pairs than in randomly permuted pairs, indicating an association between these dust and stool communities. These associations could indicate a role for the indoor environment in shaping the nascent gut microbiota, but future studies will be needed to confirm that our findings do not solely reflect a reverse pathway. Although pet ownership was strongly associated with the presence of certain genera in the dust for dogs (Agrococcus, Carnobacterium, Exiguobacterium, Herbaspirillum, Leifsonia and Neisseria) and cats (Escherichia), no clear patterns were observed in the NMDS-resolved stool community profiles as a function of pet ownership.

Comparison of three typing methods for Pseudomonas aeruginosa isolates from patients with cystic fibrosis.

The aim of this study was to compare two traditional pattern matching techniques, pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD), with the more reproducible technique of multilocus sequence typing (MLST) to genotype a blinded sample of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients. A blinded sample of 48 well-characterized CF P. aeruginosa isolates was genotyped by PFGE, RAPD, and MLST, each performed in a different laboratory. The discriminatory power and congruence between the methods were compared using the Simpson's index, Rand index, and Wallace coefficient. PFGE and MLST had the greatest congruence with the highest Rand index (0.697). The discriminatory power of PFGE, RAPD, and MLST were comparable, with high Simpson's indices (range 0.973-0.980). MLST identified the most clonal relationships. When clonality was defined as agreement between two or more methods, MLST had the greatest predictive value (100 %) in labeling strains as unique, while PFGE had the greatest predictive value (96 %) in labeling strains as clonal. This study demonstrated the highest level of agreement between PFGE and MLST in genotyping P. aeruginosa isolates from CF patients. MLST had the greatest predictive value in identifying strains as unique and, thus, has the potential to be a cost-efficient, high-throughput, first-pass typing method.

Functional analysis of the type III effectors AvrRpt2 and AvrRpm1 of Pseudomonas syringae with the use of a single-copy genomic integration system.

Gram-negative phytopathogenic bacteria require a type III secretion apparatus for pathogenesis, presumably to deliver Avr effector proteins directly into plant cells. To extend previous studies of Avr effectors that employed plasmids encoding Avr proteins, we developed a system that permits the integration of any gene into the Pseudomonas syringae genome in single copy. With this system, we confirmed earlier findings showing that P. syringae pv. maculicola strain PsmES4326 expressing the AvrRpt2 effector induces a resistance response in plants with the cognate R gene, RPS2. Chromosomally located avrRpt2, however, provoked a stronger resistance response than that observed with plasmid-expressed AvrRpt2 in RPS2+ plants. Additionally, chromosomal expression of AvrRpt2 conferred a fitness advantage on P. syringae grown in rps2- plants, aiding in growth within leaves and escape to leaf surfaces that was difficult to detect with plasmid-borne avrRpt2. Finally, with the use of the genomic integration system, we found that a chimeric protein composed of the N terminus of the heterologous AvrRpml effector and the C-terminal effector region of AvrRpt2 was delivered to plant cells. Because the C terminus of AvrRpt2 cannot translocate into plant cells on its own, this indicates that the N-terminal region can direct secretion and translocation during an infection, which supports the view that Avr proteins have a modular design. This work establishes a readily manipulatable system to study type III effectors in a biologically realistic context.

The gain-of-function Arabidopsis acd6 mutant reveals novel regulation and function of the salicylic acid signaling pathway in controlling cell death, defenses, and cell growth.

We isolated a dominant gain-of-function Arabidopsis mutant, accelerated cell death 6 (acd6), with elevated defenses, patches of dead and enlarged cells, reduced stature, and increased resistance to Pseudomonas syringae. The acd6-conferred phenotypes are suppressed by removing a key signaling molecule, salicylic acid (SA), by using the nahG transgene, which encodes SA hydroxylase. This suppression includes phenotypes that are not induced by application of SA to wild-type plants, indicating that SA acts with a second signal to cause many acd6-conferred phenotypes. acd6-nahG plants show hyperactivation of all acd6-conferred phenotypes after treatment with a synthetic inducer of the SA pathway, benzo(1,2, 3)thiadiazole-7-carbothioic acid (BTH), suggesting that SA acts with and also modulates the levels and/or activity of the second defense signal. acd6 acts partially through a NONEXPRESSOR OF PR 1 (NPR1) gene-independent pathway that activates defenses and confers resistance to P. syringae. Surprisingly, BTH-treated acd6-nahG plants develop many tumor-like abnormal growths, indicating a possible role for SA in modulating cell growth.

An X-linked gene with a degenerate Y-linked homologue in a dioecious plant.

Most flowering plants are hermaphroditic, having flowers with both male and female parts. Less than 4% of plant species are dioecious (with individuals of separate sexes), and many of these species have chromosome-mediated sex determination. The taxonomic distribution of separate sexes and chromosomal sex-determination systems in the flowering plants indicates that plant sex chromosomes have evolved recently through replicated, independent events, contrasting with the ancient origins of mammalian and insect sex chromosomes. Plant sex chromosomes, therefore, offer opportunities to study the most interesting early stages of the evolution of sex chromosomes. Here we show that a gene encoding a male-specific protein is linked to the X chromosome in the dioecious plant Silene latifolia, and that it has a degenerate homologue in the non-pairing region of the Y chromosome. The Y-linked locus has degenerated as a result of nucleotide deletion and the accumulation of repetitive sequences. We have identified both the first X-linked gene and the first pair of homologous sex-linked loci to be found in plants. The homology between the active X-linked locus and the degenerate Y-linked locus supports a common ancestry for these two loci.

Lack of degeneration of loci on the neo-Y chromosome of Drosophila americana americana.

The extent of genetic degeneration of the neo-Y chromosome of Drosophila americana americana has been investigated. Three loci, coding for the enzymes enolase, phosphoglycerate kinase and alcohol dehydrogenase, have been localized to chromosome 4 of D. a. americana, which forms the neo-Y and neo-X chromosomes. Crosses between D.a. americana and D. virilis or D. montana showed that the loci coding for these enzymes carry active alleles on the neo-Y chromosome in all wild-derived strains of americana that were tested. Intercrosses between a genetically marked stock of virilis and strains of americana were carried out, creating F3 males that were homozygous for sections of the neo-Y chromosome. The sex ratios in the F3 generation of the intercrosses showed that no lethal alleles have accumulated on any of the neo-Y chromosomes tested. There was evidence for more minor reductions in fitness, but this seems to be mainly caused by deleterious alleles that are specific to each strain. A similar picture was provided by examination of the segregation ratios of two marker genes among the F3 progeny. Overall, the data suggest that the neo-Y chromosome has undergone very little degeneration, certainly not to the extent of having lost the functions of vital genes. This is consistent with the recent origin of the neo-Y and neo-X chromosomes, and the slow rates at which the forces that cause Y chromosome degeneration are likely to work.

Seeing selection in S allele sequences.

New data on allelic sequence diversity in natural populations provide evidence for natural selection acting on the self-incompatibility loci of two plant species; there are interesting parallels with, and differences from, other polymorphic systems such as mammalian MHC loci.

Recombination and clonality in natural populations of Escherichia coli.

Bacteria such as Escherichia coli have been commonly viewed as being primarily clonal organisms. As such, the genetic variation within clones was thought to be almost exclusively the result of the mutational process. This conclusion has recently been challenged by data from DNA sequencing studies of natural isolates that are incompatible with a primarily clonal structure. Molecular population genetic analyses of these data, including gene genealogical comparisons, have raised the possibility of a much more complex population structure that may encompass relatively frequent recombination, recurrent selective sweeps and extensive ecological population subdivision.

Multiple infections of Ixodes scapularis ticks by Borrelia burgdorferi as revealed by single-strand conformation polymorphism analysis.

The genetic heterogeneity of the spirochete Borrelia burgdorferi within single adult black-legged ticks from Shelter Island, N.Y., was determined by cold, single-strand conformation polymorphism (SSCP) analysis. The central region of the ospA gene of B. burgdorferi from infected ticks was amplified by nested PCR. Amplified product of the correct size was obtained from 20 to 45 ticks (44%). This is the fraction of ticks that is expected to be infected with B. burgdorferi. Four variant classes were determined by SSCP analysis. Eight ticks were infected with a single variant, nine ticks were infected with two variants, two ticks were infected with three variants, and one tick was infected with all four variants. DNA from each variant was sequenced. Five different sequences were found. The sequence of each variant was different from that of another variant by a single base. SSCP analysis could distinguish three of the four single-base changes found in the region.

Detecting selective sweeps in naturally occurring Escherichia coli.

The nucleotide sequences of the gapA and pabB genes (separated by approximately 32.5 kb) were determined in 12 natural isolates of Escherichia coli. Three analyses were performed on the data. First, the levels of polymorphism at the loci were compared within and between E. coli and Salmonella strains relative to their degrees of constraint. Second, the gapA and pabB loci were analyzed by the Hudson-Kreitman-Aguadé (HKA) test for selective neutrality. Four additional dispersed genes (crr, putP, trp and gnd) were added to the analysis to provide the necessary frame of reference. Finally, the gene genealogies of gapA and pabB were examined for topological consistency within and between the loci. These lines of evidence indicate that some evolutionary event has recently purged the variability in the region surrounding the gapA and pabB loci in E. coli. This can best be explained by the spread of a selected allele through the global E. coli population by directional selection and the resulting loss in variability in the surrounding regions due to genetic hitchhiking.

Clonal divergence in Escherichia coli as a result of recombination, not mutation.

Nucleotide sequence analysis was performed on 12 natural isolates of Escherichia coli in four loci located in close proximity on the chromosome. A comparison of gene genealogies indicated that three recombination events have occurred in a subset of the strains (ECOR group A) in the time since their divergence from a common ancestor, while during the same time, no mutational divergence has occurred. The common ancestor of this subset existed no more than 2400 years ago, and recombination was shown to occur at a rate of 5.0 x 10(-9) changes per nucleotide per generation--50-fold higher than the mutation rate. Thus, recombination has been the dominant force driving the clonal divergence of the ECOR group A strains and must be considered a significant factor in structuring E. coli populations.