Binding of GTP to BifA is required for the production of Pel-dependent biofilms in Pseudomonas aeruginosa.

The Pel exopolysaccharide is one of the most mechanistically conserved and phylogenetically diverse bacterial biofilm matrix determinants. Pel is a major contributor to the structural integrity of Pseudomonas aeruginosa biofilms, and its biosynthesis is regulated by the binding of cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP) to the PelD receptor. c-di-GMP is synthesized from two molecules of guanosine triphosphate (GTP) by diguanylate cyclases with GGDEF domains and degraded by phosphodiesterases with EAL or HD-GYP domains. As the P. aeruginosa genome encodes 43 c-di-GMP metabolic enzymes, one way signaling specificity can be achieved is through direct interaction between specific enzyme-receptor pairs. Here, we show that the inner membrane hybrid GGDEF-EAL enzyme, BifA, directly interacts with PelD via its cytoplasmic HAMP, GGDEF, and EAL domains. Despite having no catalytic function, the degenerate active site motif of the BifA GGDEF domain (GGDQF) has retained the ability to bind GTP with micromolar affinity. Mutations that abolish GTP binding result in increased biofilm formation but stable global c-di-GMP levels. Our data suggest that BifA forms a dimer in solution and that GTP binding induces conformational changes in dimeric BifA that enhance the BifA-PelD interaction and stimulate its phosphodiesterase activity, thus reducing c-di-GMP levels and downregulating Pel biosynthesis. Structural comparisons between the dimeric AlphaFold2 model of BifA and the structures of other hybrid GGDEF-EAL proteins suggest that the regulation of BifA by GTP may occur through a novel mechanism.IMPORTANCEc-di-GMP is the most common cyclic dinucleotide used by bacteria to regulate phenotypes such as motility, biofilm formation, virulence factor production, cell cycle progression, and cell differentiation. While the identification and initial characterization of c-di-GMP metabolic enzymes are well established, our understanding of how these enzymes are regulated to provide signaling specificity remains understudied. Here we demonstrate that the inactive GGDEF domain of BifA binds GTP and regulates the adjacent phosphodiesterase EAL domain, ultimately downregulating Pel-dependent P. aeruginosa biofilm formation through an interaction with PelD. This discovery adds to the growing body of literature regarding how hybrid GGDEF-EAL enzymes are regulated and provides additional precedence for studying how direct interactions between c-di-GMP metabolic enzymes and effectors result in signaling specificity.

Evidence for the Type IV Pilus Retraction Motor PilT as a Component of the Surface Sensing System in Pseudomonas aeruginosa.

Biofilm formation begins when bacteria contacting a surface induce cellular changes to become better adapted for surface growth. One of the first changes to occur for Pseudomonas aeruginosa after surface contact is an increase in the nucleotide second messenger 3',5'-cyclic AMP (cAMP). It has been demonstrated that this increase in intracellular cAMP is dependent on functional type IV pili (T4P) relaying a signal to the Pil-Chp system, but the mechanism by which this signal is transduced remains poorly understood. Here, we investigate the role of the type IV pilus retraction motor PilT in sensing a surface and relaying that signal to cAMP production. We show that mutations in PilT, and in particular those impacting the ATPase activity of this motor protein, reduce surface-dependent cAMP production. We identify a novel interaction between PilT and PilJ, a member of the Pil-Chp system, and propose a new model whereby P. aeruginosa uses its PilT retraction motor to sense a surface and to relay that signal via PilJ to increased production of cAMP. We discuss these findings in light of current T4P-dependent surface sensing models for P. aeruginosa. IMPORTANCE T4P are cellular appendages that allow P. aeruginosa to sense a surface, leading to the production of cAMP. This second messenger not only activates virulence pathways but leads to further surface adaptation and irreversible attachment of cells. Here, we demonstrate the importance of the retraction motor PilT in surface sensing. We also present a new surface sensing model in P. aeruginosa whereby the T4P retraction motor PilT senses and transmits the surface signal, likely via its ATPase domain and interaction with PilJ, to mediate production of the second messenger cAMP.

Evidence for the Type IV Pili Retraction Motor PilT as a Component of the Surface Sensing System in Pseudomonas aeruginosa.

Biofilm formation begins when bacteria contacting a surface induce cellular changes to become better adapted for surface growth. One of the first changes to occur for Pseudomonas aeruginosa after surface contact is an increase in the nucleotide second messenger 3',5'-cyclic adenosine monophosphate (cAMP). It has been demonstrated that this increase in intracellular cAMP is dependent on functional Type IV pili (T4P) relaying a signal to the Pil-Chp system, but the mechanism by which this signal is transduced remains poorly understood. Here, we investigate the role of the Type IV pili retraction motor PilT in sensing a surface and relaying that signal to cAMP production. We show that mutations affecting the structure of PilT and in particular ATPase activity of this motor protein, reduce surface-dependent cAMP production. We identify a novel interaction between PilT and PilJ, a member of the Pil-Chp system, and propose a new model whereby P. aeruginosa uses its retraction motor to sense a surface and to relay that signal via PilJ to increased production of cAMP. We discuss these findings in light of current TFP-dependent surface sensing models for P. aeruginosa . Importance: T4P are cellular appendages that allow P. aeruginosa to sense a surface leading to the production of cAMP. This second messenger not only activates virulence pathways but leads to further surface adaptation and irreversible attachment of cells. Here, we demonstrate the importance of the retraction motor PilT in surface sensing. We also present a new surface sensing model in P. aeruginosa whereby the T4P retraction motor PilT senses and transmits the surface signal, likely via its ATPase domain and interaction with PilJ, to mediate production of the second messenger cAMP.

Biofilm Maintenance as an Active Process: Evidence that Biofilms Work Hard to Stay Put.

Biofilm formation represents a critical strategy whereby bacteria can tolerate otherwise damaging environmental stressors and antimicrobial insults. While the mechanisms bacteria use to establish a biofilm and disperse from these communities have been well-studied, we have only a limited understanding of the mechanisms required to maintain these multicellular communities. Indeed, until relatively recently, it was not clear that maintaining a mature biofilm could be considered an active, regulated process with dedicated machinery. Using Pseudomonas aeruginosa as a model system, we review evidence from recent studies that support the model that maintenance of these persistent, surface-attached communities is indeed an active process. Biofilm maintenance mechanisms include transcriptional regulation and second messenger signaling (including the production of extracellular polymeric substances). We also discuss energy-conserving pathways that play a key role in the maintenance of these communities. We hope to highlight the need for further investigation to uncover novel biofilm maintenance pathways and suggest the possibility that such pathways can serve as novel antibiofilm targets.

Pseudomonas aeruginosa Uses c-di-GMP Phosphodiesterases RmcA and MorA To Regulate Biofilm Maintenance.

While the early stages of biofilm formation have been well characterized, less is known about the requirements for Pseudomonas aeruginosa to maintain a mature biofilm. We utilized a P. aeruginosa-phage interaction to identify rmcA and morA, two genes which encode bis-(3',5')-cyclic dimeric GMP (c-di-GMP)-degrading phosphodiesterases (PDEs) and are important for the regulation of biofilm maintenance. Deletion of these genes initially results in an elevated biofilm phenotype characterized by increased production of c-di-GMP, Pel polysaccharide, and/or biofilm biomass. In contrast to the wild-type strain, these mutants were unable to maintain the biofilm when exposed to carbon-limited conditions. The susceptibility to nutrient limitation, as well as subsequent loss of biofilm viability of these mutants, was phenotypically reproduced with a stringent response mutant (ΔrelA ΔspoT), indicating that the ΔrmcA and ΔmorA mutants may be unable to appropriately respond to nutrient limitation. Genetic and biochemical data indicate that RmcA and MorA physically interact with the Pel biosynthesis machinery, supporting a model whereby unregulated Pel biosynthesis contributes to the death of the ΔrmcA and ΔmorA mutant strains in an established biofilm under nutrient limitation. These findings provide evidence that c-di-GMP-mediated regulation is required for mature biofilms of P. aeruginosa to effectively respond to changing availability of nutrients. Furthermore, the PDEs involved in biofilm maintenance are distinct from those required for establishing a biofilm, suggesting that a wide variety of c-di-GMP metabolizing enzymes in organisms such as P. aeruginosa allows for discrete control over the formation, maintenance or dispersion of biofilms.IMPORTANCE Recent advances in our understanding of c-di-GMP signaling have provided key insights into the regulation of biofilms. Despite an improved understanding of how biofilms initially form, the processes that facilitate the long-term maintenance of these multicellular communities remain opaque. We found that P. aeruginosa requires two phosphodiesterases, RmcA and MorA, to maintain a mature biofilm and that biofilms lacking these PDEs succumb to nutrient limitation and die. The biofilm maintenance deficiency observed in ΔrmcA and ΔmorA mutants was also found in the stringent response-defective ΔrelA ΔspoT strain, suggesting that a regulatory intersection between c-di-GMP signaling, extracellular polysaccharide biosynthesis, and the nutrient limitation response is important for biofilm persistence. We uncover components of an important regulatory system needed for P. aeruginosa biofilms to persist in nutrient-poor conditions and provide some of the first evidence that maintaining a mature biofilm is an active process.

The varus cemented femoral stem in total hip arthroplasty: Predictors, implications and The Femoral Access Ratio.

INTRODUCTION: We describe a novel morphological ratio, the Femoral Access Ratio, in the preoperative femur to investigate the predictors of femoral stem malalignment. METHODS: We reviewed 70 cemented femoral stems. Preoperative 'FAR' score was measured. Postoperative coronal stem alignment was measured and ten year survivorship and functional outcomes investigated. RESULTS: There were three predictors of varus stem malalignment-BMI, GT-height and 'FAR' score. Increasing BMI led to higher rates of malalignment (p = 0.048). 'FAR' score <1 lead to 68.4% of varus stems. GT height contributed most to the prediction of varus stem malalignment (p = 0.013). CONCLUSION: FAR score is a simple preoperative radiographic measurement that can predict the likelihood of femoral stem varus malalignment in cemented femoral stems.

Partial Articular Resurfacing Secondary to Pediatric Hip Chondroblastoma Curettage with a 5-YearFollow-Up: A Case Report.

CASE: This report describes a case of pediatric femoral head chondroblastoma, which was initially treated by minimally invasive curettage. At the 18-month follow-up, a subsequent osteochondral defect occurred, which was treated with a partial articular resurfacing system. At 5.5 years follow-up, he was symptom-free with minor degenerative x-ray changes. CONCLUSION: We describe a technique of focal anatomic hip resurfacing using the HemiCAP system in a pediatric chondroblastoma patient who presented with an osteochondral defect after primary curettage. This approach yielded good short-term to midterm results and is a potential alternative to total hip arthroplasty in young patients.

Prognostic Importance of Pathological Fractures in Osteosarcomas.

Aims To investigate whether pathological fractures impact on osteosarcoma patient prognosis in Ireland. Methods This was a retrospective study over 22 years in a National Orthopaedic Oncology Centre. There were 117 nonfracture cases and 15 fracture cases. Outcome measures included 5 and 10 year event-free (EFS) and overall survival (OS). Kaplan-Meier curves assessed length of survival and time to death. Results Pathological fracture has no significant effect on 10 year EFS or 10 year OS. 3 factors strongly associate with 10 year OS rates: American Joint Committee on Cancer (AJCC) classification (p<0.001), Metastases site (p<0.001) and Distant recurrence (p<0.001). Fractures had poorer post-chemotherapeutic necrosis rates (p=0.005). Conclusion Pathological fractures have no significant effect on survival rates or length of survival in an Irish population. The effect of pathological fractures on necrosis rates must be explored in future research.

Comparison of the compressive mechanical properties of auricular and costal cartilage from patients with microtia.

Children born with a small or absent ears undergo surgical reconstruction to restore their auricle. Currently, rib (costal) cartilage is used to carve the auricle. However as alternative, tissue engineered and synthetic materials are being developed to restore the auricle shape to overcome donor site morbidity and limited availability of rib cartilage. However, to date there is limited knowledge regarding the mechanical properties of the auricular and costal cartilage to optimise the required compressive properties of the graft. The remnant auricular and costal cartilage from 20 patients undergoing stage-1 microtia surgery was harvested. On the day of surgery, the cartilage was evaluated in compression, with each sample loaded to 300 g at 1 mm/s. RESULTS: The costal cartilage was observed to have a significantly higher Young's Elastic Modulus than auricular cartilage (average costal cartilage 11.43 MPa vs average auricular cartilage 2 MPa, p < 0.0001). The auricular cartilage showed a significantly higher relaxation rate than costal cartilage (average costal cartilage 0.72 MPa10-4 vs average auricular cartilage 1.93 MPa10-4, p < 0.05). The final absolute relaxation was significantly lower for elastic cartilage than costal cartilage (average costal cartilage 3.35 MPa vs average auricular cartilage 0.2 MPa, p < 0.0001). Alloplastic cartilage replacements used as alternatives for reconstruction were also evaluated. Silicone, Gore-Tex and Medpor were observed to have significantly higher Young's Elastic Modulus than costal and auricular cartilage. Costal cartilage has a higher Young's Elastic Modulus in compression compared to auricular cartilage. Current synthetic materials used to replace synthetic cartilage do not mimic costal cartilage, which should be addressed in the future.

Osteosarcoma in a Patient Receiving Long-Term TNF Inhibitor Therapy.

Patients with inflammatory bowel disease (IBD) have an increased risk of developing malignancy. The use of immunosuppressive therapies and tumour necrosis factor (TNF) inhibitors in these patients may provide a further risk for the development of malignancy. We report the clinical and pathological findings of a high grade osteosarcoma in a patient with Crohns disease receiving TNF inhibitor therapy. In this case, a 32-year old female presented with a painful right knee after receiving maintenance adalimumab for Crohns disease for a period of six years. There is a substantial hypothetical link between TNF inhibitor regimens such as adalimumab and an increased risk of malignancy. TNF inhibitor therapy should be ceased and chemotherapy and surgery is an effective combined modality approach in these patients. The role of TNF inhibitors in patients after cancer diagnosis is uncertain and further research is required to assess efficacy and safety.

Perioperative chemotherapy in the treatment of osteosarcoma: a 26-year single institution review.

BACKGROUND: Chemotherapy in the multimodality treatment of osteosarcoma has improved survival. Reported outcomes on adult patients are limited. Poor necrosis rates post neoadjuvant chemotherapy (NAC) is considered an adverse prognostic factor and attempts have been made to improve survival in this group. PATIENTS AND METHODS: Adult and young adult patients diagnosed with osteosarcoma between January 1986 and August 2012 were retrospectively reviewed. Patients identified were stratified according to stage (localised or metastatic) and age (≤40 and >40 years). Event free survival (EFS) and overall survival (OS) outcomes were determined. In patients with localised disease ≤40 years, survival was assessed according to necrosis rates post NAC (<90 and ≥90%). NAC consisted of two cycles of methotrexate alternating with doxorubicin/cisplatin (MAP) followed by definitive surgery. Those with ≥90% tumour necrosis continued on MAP. Patients with <90% necrosis received ifosfamide and etoposide (IE) post operatively. RESULTS: A total of 108 patients were reviewed and 97 were included. Median age was 23 years (range 16-75) and 70% of patients were male. Five year EFS and OS across all groups was 57% and 63% respectively. Of the patients with localised disease (N = 81), 5-year overall survival (OS), with a median follow up of 7 years (2-26) was 70% (p < 0.0001). Patients aged 16-40 (N = 68) with localised osteosarcoma had a significantly improved 5-year OS (74%) compared to those >40 years (N = 13) (42%) (p = 0.004). Of the 68 patients with localised osteosarcoma ≤40 years, 62 were evaluated according to necrosis rates post MAP. In 33 patients who achieved ≥90% necrosis and continued MAP, 5-year OS was 82%. In 29 patients who had <90% tumour necrosis and received adjuvant IE, 5-year OS was 68% (p = 0.15). Multivariate analysis confirmed age and stage as prognostic factors but not poor necrosis rates in our treated population. CONCLUSIONS: Long-term survival outcomes in a predominantly adult Irish population are similar to large reported trials. Age and stage at diagnosis are prognostic. Postoperative ifosfamide/etoposide alone in patients with poor necrosis rates is a feasible regimen, but its role in the adjuvant setting remains uncertain.

Cyclic di-GMP-mediated repression of swarming motility by Pseudomonas aeruginosa PA14 requires the MotAB stator.

The second messenger cyclic diguanylate (c-di-GMP) plays a critical role in the regulation of motility. In Pseudomonas aeruginosa PA14, c-di-GMP inversely controls biofilm formation and surface swarming motility, with high levels of this dinucleotide signal stimulating biofilm formation and repressing swarming. P. aeruginosa encodes two stator complexes, MotAB and MotCD, that participate in the function of its single polar flagellum. Here we show that the repression of swarming motility requires a functional MotAB stator complex. Mutating the motAB genes restores swarming motility to a strain with artificially elevated levels of c-di-GMP as well as stimulates swarming in the wild-type strain, while overexpression of MotA from a plasmid represses swarming motility. Using point mutations in MotA and the FliG rotor protein of the motor supports the conclusion that MotA-FliG interactions are critical for c-di-GMP-mediated swarming inhibition. Finally, we show that high c-di-GMP levels affect the localization of a green fluorescent protein (GFP)-MotD fusion, indicating a mechanism whereby this second messenger has an impact on MotCD function. We propose that when c-di-GMP level is high, the MotAB stator can displace MotCD from the motor, thereby affecting motor function. Our data suggest a newly identified means of c-di-GMP-mediated control of surface motility, perhaps conserved among Pseudomonas, Xanthomonas, and other organisms that encode two stator systems.

Extra-abdominal desmoid tumours: a review of the literature.

Extra-abdominal desmoid lesions, otherwise known as aggressive fibromatosis, are slow-growing benign lesions which may be encountered in clinical practice. Recent controversies exist regarding their optimal treatment. Given their benign nature, is major debulking surgery justified, or is it worth administering chemotherapy for a disease process which unusually defies common teaching and responds to such medications? We present a literature review of this particular pathology discussing the aetiology, clinical presentation, and various current controversies in the treatment options.

Minor pilins of the type IV pilus system participate in the negative regulation of swarming motility.

Pseudomonas aeruginosa exhibits distinct surface-associated behaviors, including biofilm formation, flagellum-mediated swarming motility, and type IV pilus-driven twitching. Here, we report a role for the minor pilins, PilW and PilX, components of the type IV pilus assembly machinery, in the repression of swarming motility. Mutating either the pilW or pilX gene alleviates the inhibition of swarming motility observed for strains with elevated levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP) due to loss of BifA, a c-di-GMP-degrading phosphodiesterase. Blocking PilD peptidase-mediated processing of PilW and PilX renders the unprocessed proteins defective for pilus assembly but still functional in c-di-GMP-mediated swarming repression, indicating our ability to separate these functions. Strains with mutations in pilW or pilX also fail to exhibit the increase in c-di-GMP levels observed when wild-type (WT) or bifA mutant cells are grown on a surface. We also provide data showing that c-di-GMP levels are increased upon PilY1 overexpression in surface-grown cells and that this c-di-GMP increase does not occur in the absence of the SadC diguanylate cyclase. Increased levels of endogenous PilY1, PilX, and PilA are observed when cells are grown on a surface compared to liquid growth, linking surface growth and enhanced signaling via SadC. Our data support a model wherein PilW, PilX, and PilY1, in addition to their role(s) in type IV pilus biogenesis, function to repress swarming via modulation of intracellular c-di-GMP levels. By doing so, these pilus assembly proteins contribute to P. aeruginosa's ability to coordinately regulate biofilm formation with its two surface motility systems.

Serial analysis of the gut and respiratory microbiome in cystic fibrosis in infancy: interaction between intestinal and respiratory tracts and impact of nutritional exposures.

UNLABELLED: Pulmonary damage caused by chronic colonization of the cystic fibrosis (CF) lung by microbial communities is the proximal cause of respiratory failure. While there has been an effort to document the microbiome of the CF lung in pediatric and adult patients, little is known regarding the developing microflora in infants. We examined the respiratory and intestinal microbiota development in infants with CF from birth to 21 months. Distinct genera dominated in the gut compared to those in the respiratory tract, yet some bacteria overlapped, demonstrating a core microbiota dominated by Veillonella and Streptococcus. Bacterial diversity increased significantly over time, with evidence of more rapidly acquired diversity in the respiratory tract. There was a high degree of concordance between the bacteria that were increasing or decreasing over time in both compartments; in particular, a significant proportion (14/16 genera) increasing in the gut were also increasing in the respiratory tract. For 7 genera, gut colonization presages their appearance in the respiratory tract. Clustering analysis of respiratory samples indicated profiles of bacteria associated with breast-feeding, and for gut samples, introduction of solid foods even after adjustment for the time at which the sample was collected. Furthermore, changes in diet also result in altered respiratory microflora, suggesting a link between nutrition and development of microbial communities in the respiratory tract. Our findings suggest that nutritional factors and gut colonization patterns are determinants of the microbial development of respiratory tract microbiota in infants with CF and present opportunities for early intervention in CF with altered dietary or probiotic strategies. IMPORTANCE: While efforts have been focused on assessing the microbiome of pediatric and adult cystic fibrosis (CF) patients to understand how chronic colonization by these microbes contributes to pulmonary damage, little is known regarding the earliest development of respiratory and gut microflora in infants with CF. Our findings suggest that colonization of the respiratory tract by microbes is presaged by colonization of the gut and demonstrated a role of nutrition in development of the respiratory microflora. Thus, targeted dietary or probiotic strategies may be an effective means to change the course of the colonization of the CF lung and thereby improve patient outcomes.

Prevalence of streptococci and increased polymicrobial diversity associated with cystic fibrosis patient stability.

Diverse microbial communities chronically colonize the lungs of cystic fibrosis patients. Pyrosequencing of amplicons for hypervariable regions in the 16S rRNA gene generated taxonomic profiles of bacterial communities for sputum genomic DNA samples from 22 patients during a state of clinical stability (outpatients) and 13 patients during acute exacerbation (inpatients). We employed quantitative PCR (qPCR) to confirm the detection of Pseudomonas aeruginosa and Streptococcus by the pyrosequencing data and human oral microbe identification microarray (HOMIM) analysis to determine the species of the streptococci identified by pyrosequencing. We show that outpatient sputum samples have significantly higher bacterial diversity than inpatients, but maintenance treatment with tobramycin did not impact overall diversity. Contrary to the current dogma in the field that Pseudomonas aeruginosa is the dominant organism in the majority of cystic fibrosis patients, Pseudomonas constituted the predominant genera in only half the patient samples analyzed and reported here. The increased fractional representation of Streptococcus in the outpatient cohort relative to the inpatient cohort was the strongest predictor of clinically stable lung disease. The most prevalent streptococci included species typically associated with the oral cavity (Streptococcus salivarius and Streptococcus parasanguis) and the Streptococcus milleri group species. These species of Streptococcus may play an important role in increasing the diversity of the cystic fibrosis lung environment and promoting patient stability.

Whipple's procedure for an oligometastasis to the pancreas from a leiomyosarcoma of the thigh.

BACKGROUND: Pancreatic tumours are most frequently primary, with lesions secondary to metastasis uncommon. METHODS: This report describes the case of a 61-year-old man who underwent resection of a right thigh leiomyosarcoma 2 years prior to presentation with obstructive jaundice. Subsequent CT and endoscopic ultrasound (EUS) diagnosed metastatic leiomyosarcoma to the pancreatic head for which he underwent a Whipple's pancreaticoduodenectomy. CONCLUSION: Metastasis from an extremity leiomyosarcoma to the pancreas is an extremely rare entity, which can be diagnosed by EUS and treated successfully by pancreaticoduodenectomy.

Prevalence, conservation and functional analysis of Yersinia and Escherichia CRISPR regions in clinical Pseudomonas aeruginosa isolates.

Here, we report the characterization of 122 Pseudomonas aeruginosa clinical isolates from three distinct geographical locations: Dartmouth Hitchcock Medical Center in New Hampshire, USA, the Charles T. Campbell Eye Microbiology Lab at the University of Pittsburgh Medical Center, USA, and the Aravind Eye Hospital in Madurai, India. We identified and located clustered regularly interspaced short palindromic repeats (CRISPR) in 45/122 clinical isolates and sequenced these CRISPR, finding that Yersinia subtype CRISPR regions (33 %) were more prevalent than the Escherichia CRISPR region subtype (6 %) in these P. aeruginosa clinical isolates. Further, we observed 132 unique spacers from these 45 CRISPR that are 100 % identical to prophages or sequenced temperate bacteriophage capable of becoming prophages. Most intriguingly, all of these 132 viral spacers matched to temperate bacteriophage/prophages capable of inserting into the host chromosome, but not to extrachromosomally replicating lytic P. aeruginosa bacteriophage. We next assessed the ability of the more prevalent Yersinia subtype CRISPR regions to mediate resistance to bacteriophage infection or lysogeny by deleting the entire CRISPR region from sequenced strain UCBPP-PA14 and six clinical isolates. We found no change in CRISPR-mediated resistance to bacteriophage infection or lysogeny rate even for CRISPR with spacers 100 % identical to a region of the infecting bacteriophage. Lastly, to show these CRISPR and cas genes were expressed and functional, we demonstrated production of small CRISPR RNAs. This work provides both the first examination to our knowledge of CRISPR regions within clinical P. aeruginosa isolates and a collection of defined CRISPR-positive and -negative strains for further CRISPR and cas gene studies.