A Standardized Approach To Evaluation And Reporting of Synovial Histopathology In Two Surgically Induced Murine Models Of Osteoarthritis.

OBJECTIVE: Synovial pathology has been linked to osteoarthritis (OA) pain in patients. Microscopic grading systems for synovial changes in human OA have been described, but a standardized approach for murine models of OA is needed. We sought to develop a reproducible approach and set of minimum recommendations for reporting of synovial histopathology in mouse models of OA. METHODS: Coronal and sagittal sections from male mouse knee joints subjected to destabilization of medial meniscus (DMM) or partial meniscectomy (PMX) were collected as part of other studies. Stains included Hematoxylin and Eosin (H&E), Toluidine Blue (T-Blue) and Safranin O/Fast Green (Saf-O). Four blinded readers graded pathological features (hyperplasia, cellularity, and fibrosis) at specific anatomic locations. Inter-reader agreement of each feature score was determined. RESULTS: There was acceptable to very good agreement when using 3-4 individual readers. After DMM and PMX, expected medial predominant changes in hyperplasia and cellularity were observed, with fibrosis noted at 12 weeks post-PMX. Synovial changes were consistent from section to section in the mid-joint area. When comparing stains, H&E and T-blue resulted in better agreement compared to Saf-O stain. CONCLUSIONS: To account for the pathologic and anatomic variability in synovial pathology and allow for a more standardized evaluation that can be compared across studies, we recommend evaluating a minimum set of 3 pathological features at standardized anatomic areas. Further, we suggest reporting individual feature scores separately before relying on a single summed "synovitis" score. H&E or T-blue are preferred, inter-reader agreement for each feature should be considered.

Yinchen gongying decoction mitigates CCl4-induced chronic liver injury and fibrosis in mice implicated in inhibition of the FoxO1/TGF-ß1/ Smad2/3 and YAP signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Liver fibrosis (LF) is a common reversible consequence of chronic liver damage with limited therapeutic options. Yinchen Gongying decoction (YGD) composed of two homologous plants: (Artemisia capillaris Thunb, Taraxacum monochlamydeum Hand.-Mazz.), has a traditionally application as a medicinal diet for acute icteric hepatitis. However, its impact on LF and underlying mechanisms remain unclear. AIM OF THE STUDY: This study aims to assess the impact of YGD on a carbon tetrachloride (CCl4) induced liver fibrosis and elucidate its possible mechanisms. The study seeks to establish an experimental foundation for YGD as a candidate drug for hepatic fibrosis. MATERIALS AND METHODS: LC-MS/MS identified 11 blood-entry components in YGD, and network pharmacology predicted their involvement in the FoxO signaling pathway, insulin resistance, and PI3K-AKT signaling pathway. Using a CCl4-induced LF mouse model, YGD's protective effects were evaluated in comparison to a positive control and a normal group. The underlying mechanisms were explored through the assessments of hepatic stellate cells (HSCs) activation, fibrotic signaling, and inflammation. RESULTS: YGD treatment significantly improved liver function, enhanced liver morphology, and reduced liver collagen deposition in CCl4-induced LF mice. Mechanistically, YGD inhibited HSC activation, elevated MMPs/TIMP1 ratios, suppressed the FoxO1/TGF-ß1/Smad2/3 and YAP pathways, and exhibited anti-inflammatory and antioxidant effects. Notably, YGD improved the insulin signaling pathway. CONCLUSION: YGD mitigates LF in mice by modulating fibrotic and inflammatory pathways, enhancing antioxidant responses, and specifically inhibiting FoxO1/TGF-ß1/Smad2/3 and YAP signal pathways.

Recommendations For a Standardized Approach to Histopathologic Evaluation of Synovial Membrane in Murine Models of Experimental Osteoarthritis.

Background: Synovial pathology has been linked to osteoarthritis (OA) pain in patients. Microscopic grading systems for synovial changes in human OA have been described, but a standardized approach for murine models of OA is needed. We sought to develop a reproducible approach and set of minimum recommendations for synovial histopathology in mouse models of OA. Methods: Coronal and sagittal sections from male mouse knee joints subjected to destabilization of medial meniscus (DMM) or partial meniscectomy (PMX) were collected as part of other studies. Stains included Hematoxylin and Eosin (H&E), Toluidine Blue (T-Blue) and Safranin O/Fast Green (Saf-O). Four blinded readers graded pathological features (hyperplasia, cellularity, and fibrosis) at specific anatomic locations in the medial and lateral compartments. Inter-reader reliability of each feature was determined. Results: There was acceptable to very good agreement between raters. After DMM, increased hyperplasia and cellularity and a trend towards increased fibrosis were observed 6 weeks after DMM in the medial locations, and persisted up to 16 weeks. In the PMX model, cellularity and hyperplasia were evident in both medial and lateral compartments while fibrotic changes were largely seen on the medial side. Synovial changes were consistent from section to section in the mid-joint area mice. H&E, T-blue, and Saf-O stains resulted in comparable reliability. Conclusions: To allow for a standard evaluation that can be implemented and compared across labs and studies, we recommend using 3 readers to evaluate a minimum set of 3 pathological features at standardized anatomic areas. Pre-defining areas to be scored, and reliability for each pathologic feature should be considered.

RIPK1-dependent apoptosis bypasses pathogen blockade of innate signaling to promote immune defense.

Many pathogens deliver virulence factors or effectors into host cells in order to evade host defenses and establish infection. Although such effector proteins disrupt critical cellular signaling pathways, they also trigger specific antipathogen responses, a process termed "effector-triggered immunity." The Gram-negative bacterial pathogen Yersinia inactivates critical proteins of the NF-κB and MAPK signaling cascade, thereby blocking inflammatory cytokine production but also inducing apoptosis. Yersinia-induced apoptosis requires the kinase activity of receptor-interacting protein kinase 1 (RIPK1), a key regulator of cell death, NF-κB, and MAPK signaling. Through the targeted disruption of RIPK1 kinase activity, which selectively disrupts RIPK1-dependent cell death, we now reveal that Yersinia-induced apoptosis is critical for host survival, containment of bacteria in granulomas, and control of bacterial burdens in vivo. We demonstrate that this apoptotic response provides a cell-extrinsic signal that promotes optimal innate immune cytokine production and antibacterial defense, demonstrating a novel role for RIPK1 kinase-induced apoptosis in mediating effector-triggered immunity to circumvent pathogen inhibition of immune signaling.

Guanylate Binding Proteins Regulate Inflammasome Activation in Response to Hyperinjected Yersinia Translocon Components.

Gram-negative bacterial pathogens utilize virulence-associated secretion systems to inject, or translocate, effector proteins into host cells to manipulate cellular processes and promote bacterial replication. However, translocated bacterial products are sensed by nucleotide binding domain and leucine-rich repeat-containing proteins (NLRs), which trigger the formation of a multiprotein complex called the inflammasome, leading to secretion of interleukin-1 (IL-1) family cytokines, pyroptosis, and control of pathogen replication. Pathogenic Yersinia bacteria inject effector proteins termed Yops, as well as pore-forming proteins that comprise the translocon itself, into target cells. The Yersinia translocation regulatory protein YopK promotes bacterial virulence by limiting hyperinjection of the translocon proteins YopD and YopB into cells, thereby limiting cellular detection of Yersinia virulence activity. How hyperinjection of translocon proteins leads to inflammasome activation is currently unknown. We found that translocated YopB and YopD colocalized with the late endosomal/lysosomal protein LAMP1 and that the frequency of YopD and LAMP1 association correlated with the level of caspase-1 activation in individual cells. We also observed colocalization between YopD and Galectin-3, an indicator of endosomal membrane damage. Intriguingly, YopK limited the colocalization of Galectin-3 with YopD, suggesting that YopK limits the induction or sensing of endosomal membrane damage by components of the type III secretion system (T3SS) translocon. Furthermore, guanylate binding proteins (GBPs) encoded on chromosome 3 (GbpChr3 ), which respond to pathogen-induced damage or alteration of host membranes, were necessary for inflammasome activation in response to hyperinjected YopB/-D. Our findings indicate that lysosomal damage by Yersinia translocon proteins promotes inflammasome activation and implicate GBPs as key regulators of this process.

Activity of Uncleaved Caspase-8 Controls Anti-bacterial Immune Defense and TLR-Induced Cytokine Production Independent of Cell Death.

Caspases regulate cell death programs in response to environmental stresses, including infection and inflammation, and are therefore critical for the proper operation of the mammalian immune system. Caspase-8 is necessary for optimal production of inflammatory cytokines and host defense against infection by multiple pathogens including Yersinia, but whether this is due to death of infected cells or an intrinsic role of caspase-8 in TLR-induced gene expression is unknown. Caspase-8 activation at death signaling complexes results in its autoprocessing and subsequent cleavage and activation of its downstream apoptotic targets. Whether caspase-8 activity is also important for inflammatory gene expression during bacterial infection has not been investigated. Here, we report that caspase-8 plays an essential cell-intrinsic role in innate inflammatory cytokine production in vivo during Yersinia infection. Unexpectedly, we found that caspase-8 enzymatic activity regulates gene expression in response to bacterial infection as well as TLR signaling independently of apoptosis. Using newly-generated mice in which caspase-8 autoprocessing is ablated (Casp8DA/DA), we now demonstrate that caspase-8 enzymatic activity, but not autoprocessing, mediates induction of inflammatory cytokines by bacterial infection and a wide variety of TLR stimuli. Because unprocessed caspase-8 functions in an enzymatic complex with its homolog cFLIP, our findings implicate the caspase-8/cFLIP heterodimer in control of inflammatory cytokines during microbial infection, and provide new insight into regulation of antibacterial immune defense.

The Effect of Total Household Decolonization on Clearance of Colonization With Methicillin-Resistant Staphylococcus aureus.

OBJECTIVE To determine the impact of total household decolonization with intranasal mupirocin and chlorhexidine gluconate body wash on recurrent methicillin-resistant Staphylococcus aureus (MRSA) infection among subjects with MRSA skin and soft-tissue infection. DESIGN Three-arm nonmasked randomized controlled trial. SETTING Five academic medical centers in Southeastern Pennsylvania. PARTICIPANTS Adults and children presenting to ambulatory care settings with community-onset MRSA skin and soft-tissue infection (ie, index cases) and their household members. INTERVENTION Enrolled households were randomized to 1 of 3 intervention groups: (1) education on routine hygiene measures, (2) education plus decolonization without reminders (intranasal mupirocin ointment twice daily for 7 days and chlorhexidine gluconate on the first and last day), or (3) education plus decolonization with reminders, where subjects received daily telephone call or text message reminders. MAIN OUTCOME MEASURES Owing to small numbers of recurrent infections, this analysis focused on time to clearance of colonization in the index case. RESULTS Of 223 households, 73 were randomized to education-only, 76 to decolonization without reminders, 74 to decolonization with reminders. There was no significant difference in time to clearance of colonization between the education-only and decolonization groups (log-rank P=.768). In secondary analyses, compliance with decolonization was associated with decreased time to clearance (P=.018). CONCLUSIONS Total household decolonization did not result in decreased time to clearance of MRSA colonization among adults and children with MRSA skin and soft-tissue infection. However, subjects who were compliant with the protocol had more rapid clearance Trial registration. ClinicalTrials.gov identifier: NCT00966446 Infect Control Hosp Epidemiol 2016;1-8.

Comparison of Culture-Based Methods for Identification of Colonization with Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus in the Context of Cocolonization.

Two screening methods to detect staphylococcal colonization in humans were compared. Direct plating to CHROMagar (BD Diagnostics) was compared to a broth preenrichment followed by plating to Baird-Parker agar. The broth-enrichment method was comparable to CHROMagar for methicillin-resistant Staphylococcus aureas (MRSA) detection, but the enrichment method was optimum for recovery of coagulase-positive Staphylococcus spp.

Risk factors for recurrent colonization with methicillin-resistant Staphylococcus aureus in community-dwelling adults and children.

OBJECTIVE To identify risk factors for recurrent methicillin-resistant Staphylococcus aureus (MRSA) colonization. DESIGN Prospective cohort study conducted from January 1, 2010, through December 31, 2012. SETTING Five adult and pediatric academic medical centers. PARTICIPANTS Subjects (ie, index cases) who presented with acute community-onset MRSA skin and soft-tissue infection. METHODS Index cases and all household members performed self-sampling for MRSA colonization every 2 weeks for 6 months. Clearance of colonization was defined as 2 consecutive sampling periods with negative surveillance cultures. Recurrent colonization was defined as any positive MRSA surveillance culture after clearance. Index cases with recurrent MRSA colonization were compared with those without recurrence on the basis of antibiotic exposure, household demographic characteristics, and presence of MRSA colonization in household members. RESULTS The study cohort comprised 195 index cases; recurrent MRSA colonization occurred in 85 (43.6%). Median time to recurrence was 53 days (interquartile range, 36-84 days). Treatment with clindamycin was associated with lower risk of recurrence (odds ratio, 0.52; 95% CI, 0.29-0.93). Higher percentage of household members younger than 18 was associated with increased risk of recurrence (odds ratio, 1.01; 95% CI, 1.00-1.02). The association between MRSA colonization in household members and recurrent colonization in index cases did not reach statistical significance in primary analyses. CONCLUSION A large proportion of patients initially presenting with MRSA skin and soft-tissue infection will have recurrent colonization after clearance. The reduced rate of recurrent colonization associated with clindamycin may indicate a unique role for this antibiotic in the treatment of such infection.

Duration of Colonization and Determinants of Earlier Clearance of Colonization With Methicillin-Resistant Staphylococcus aureus.

BACKGROUND: The duration of colonization and factors associated with clearance of methicillin-resistant Staphylococcus aureus (MRSA) after community-onset MRSA skin and soft-tissue infection (SSTI) remain unclear. METHODS: We conducted a prospective cohort study of patients with acute MRSA SSTI presenting to 5 adult and pediatric academic hospitals from 1 January 2010 through 31 December 2012. Index patients and household members performed self-sampling for MRSA colonization every 2 weeks for 6 months. Clearance of colonization was defined as negative MRSA surveillance cultures during 2 consecutive sampling periods. A Cox proportional hazards regression model was developed to identify determinants of clearance of colonization. RESULTS: Two hundred forty-three index patients were included. The median duration of MRSA colonization after SSTI diagnosis was 21 days (95% confidence interval [CI], 19-24), and 19.8% never cleared colonization. Treatment of the SSTI with clindamycin was associated with earlier clearance (hazard ratio [HR], 1.72; 95% CI, 1.28-2.30; P < .001). Older age (HR, 0.99; 95% CI, .98-1.00; P = .01) was associated with longer duration of colonization. There was a borderline significant association between increased number of household members colonized with MRSA and later clearance of colonization in the index patient (HR, 0.85; 95% CI, .71-1.01; P = .06). CONCLUSIONS: With a systematic, regular sampling protocol, duration of MRSA colonization was noted to be shorter than previously reported, although 19.8% of patients remained colonized at 6 months. The association between clindamycin and shorter duration of colonization after MRSA SSTI suggests a possible role for the antibiotic selected for treatment of MRSA infection.

The shared microbiota of humans and companion animals as evaluated from Staphylococcus carriage sites.

BACKGROUND: Staphylococcus aureus and other coagulase-positive staphylococci (CPS) colonize skin and mucous membrane sites and can cause skin and soft tissue infections (SSTIs) in humans and animals. Factors modulating methicillin-resistant S. aureus (MRSA) colonization and infection in humans remain unclear, including the role of the greater microbial community and environmental factors such as contact with companion animals. In the context of a parent study evaluating the households of outpatients with community MRSA SSTI, the objectives of this study were 1) to characterize the microbiota that colonizes typical coagulase-positive Staphylococcus spp. carriage sites in humans and their companion pets, 2) to analyze associations between Staphylococcus infection and carriage and the composition and diversity of microbial communities, and 3) to analyze factors that influence sharing of microbiota between pets and humans. RESULTS: We enrolled 25 households containing 56 pets and 30 humans. Sampling locations were matched to anatomical sites cultured by the parent study for MRSA and other CPS. Bacterial microbiota were characterized by sequencing of 16S ribosomal RNA genes. Household membership was strongly associated with microbial communities, in both humans and pets. Pets were colonized with a greater relative abundance of Proteobacteria, whereas people were colonized with greater relative abundances of Firmicutes and Actinobacteria. We did not detect differences in microbiota associated with MRSA SSTI, or carriage of MRSA, S. aureus or CPS. Humans in households without pets were more similar to each other than humans in pet-owning households, suggesting that companion animals may play a role in microbial transfer. We examined changes in microbiota over a 3-month time period and found that pet staphylococcal carriage sites were more stable than human carriage sites. CONCLUSIONS: We characterized and identified patterns of microbiota sharing and stability between humans and companion animals. While we did not detect associations with MRSA SSTI, or carriage of MRSA, S. aureus or CPS in this small sample size, larger studies are warranted to fully explore how microbial communities may be associated with and contribute to MRSA and/or CPS colonization, infection, and recurrence.

Caspase-8 mediates caspase-1 processing and innate immune defense in response to bacterial blockade of NF-κB and MAPK signaling.

Toll-like receptor signaling and subsequent activation of NF-κB- and MAPK-dependent genes during infection play an important role in antimicrobial host defense. The YopJ protein of pathogenic Yersinia species inhibits NF-κB and MAPK signaling, resulting in blockade of NF-κB-dependent cytokine production and target cell death. Nevertheless, Yersinia infection induces inflammatory responses in vivo. Moreover, increasing the extent of YopJ-dependent cytotoxicity induced by Yersinia pestis and Yersinia pseudotuberculosis paradoxically leads to decreased virulence in vivo, suggesting that cell death promotes anti-Yersinia host defense. However, the specific pathways responsible for YopJ-induced cell death and how this cell death mediates immune defense against Yersinia remain poorly defined. YopJ activity induces processing of multiple caspases, including caspase-1, independently of inflammasome components or the adaptor protein ASC. Unexpectedly, caspase-1 activation in response to the activity of YopJ required caspase-8, receptor-interacting serine/threonine kinase 1 (RIPK1), and Fas-associated death domain (FADD), but not RIPK3. Furthermore, whereas RIPK3 deficiency did not affect YopJ-induced cell death or caspase-1 activation, deficiency of both RIPK3 and caspase-8 or FADD completely abrogated Yersinia-induced cell death and caspase-1 activation. Mice lacking RIPK3 and caspase-8 in their hematopoietic compartment showed extreme susceptibility to Yersinia and were deficient in monocyte and neutrophil-derived production of proinflammatory cytokines. Our data demonstrate for the first time to our knowledge that RIPK1, FADD, and caspase-8 are required for YopJ-induced cell death and caspase-1 activation and suggest that caspase-8-mediated cell death overrides blockade of immune signaling by YopJ to promote anti-Yersinia immune defense.

Oxidative metabolism enables Salmonella evasion of the NLRP3 inflammasome.

Microbial infection triggers assembly of inflammasome complexes that promote caspase-1-dependent antimicrobial responses. Inflammasome assembly is mediated by members of the nucleotide binding domain leucine-rich repeat (NLR) protein family that respond to cytosolic bacterial products or disruption of cellular processes. Flagellin injected into host cells by invading Salmonella induces inflammasome activation through NLRC4, whereas NLRP3 is required for inflammasome activation in response to multiple stimuli, including microbial infection, tissue damage, and metabolic dysregulation, through mechanisms that remain poorly understood. During systemic infection, Salmonella avoids NLRC4 inflammasome activation by down-regulating flagellin expression. Macrophages exhibit delayed NLRP3 inflammasome activation after Salmonella infection, suggesting that Salmonella may evade or prevent the rapid activation of the NLRP3 inflammasome. We therefore screened a Salmonella Typhimurium transposon library to identify bacterial factors that limit NLRP3 inflammasome activation. Surprisingly, absence of the Salmonella TCA enzyme aconitase induced rapid NLRP3 inflammasome activation. This inflammasome activation correlated with elevated levels of bacterial citrate, and required mitochondrial reactive oxygen species and bacterial citrate synthase. Importantly, Salmonella lacking aconitase displayed NLRP3- and caspase-1/11-dependent attenuation of virulence, and induced elevated serum IL-18 in wild-type mice. Together, our data link Salmonella genes controlling oxidative metabolism to inflammasome activation and suggest that NLRP3 inflammasome evasion promotes systemic Salmonella virulence.

Colonization of Cimex lectularius with methicillin-resistant Staphylococcus aureus.

A recent paper published by Lowe and Romney in Emerging Infectious Diseases titled, Bed bugs as Vectors for Drug-Resistant Bacteria has sparked a renewed interest in bed bug vector potential. We followed a pyrethroid resistant strain of the human bed bug (Cimex lectularius, L.) fed either human blood or human blood with added methicillin resistant Staphylococcus aureus (MRSA) for 9 days post-feeding. Results indicated that while the bed bug midgut is a hospitable environment for MRSA, the bacteria does not survive longer than 9 days within the midgut. Additionally, MRSA is not amplified within the midgut of the bug as the infection was cleared within 9 days. Due to the weekly feeding behaviours of bed bugs, these results suggest that bed bug transmission of MRSA is highly unlikely.

The effect of a hospital-wide urine culture screening intervention on the incidence of extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella species.

OBJECTIVE: Optimal strategies for limiting the transmission of extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella spp (ESBL-EK) in the hospital setting remain unclear. The objective of this study was to evaluate the impact of a urine culture screening strategy on the incidence of ESBL-EK. DESIGN: Prospective quasi-experimental study. SETTING: Two intervention hospitals and one control hospital within a university health system from 2005 to 2009. PATIENTS AND INTERVENTION: All clinical urine cultures with E. coli or Klebsiella spp were screened for ESBL-EK. Patients determined to be colonized or infected with ESBL-EK were placed in a private room with contact precautions. The primary outcome of interest was nosocomial ESBL-EK incidence in nonurinary clinical cultures (cases occurring more than 48 hours after admission). Changes in monthly ESBL-EK incidence rates were evaluated with mixed-effects Poisson regression models, with adjustment for institution-level characteristics (eg, total admissions). RESULTS: The overall incidence of ESBL-EK increased from 1.42/10,000 patient-days to 2.16/10,000 patient-days during the study period. The incidence of community-acquired ESBL-EK increased nearly 3-fold, from 0.33/10,000 patient-days to 0.92/10,000 patient-days (P < .001). On multivariable analysis, the intervention was not significantly associated with a reduction in nosocomial ESBL-EK incidence (incidence rate ratio, 1.38 [95% confidence interval, 0.83-2.31]; P - .21). CONCLUSIONS: Universal screening of clinical urine cultures for ESBL-EK did not result in a reduction in nosocomial ESBL-EK incidence rates, most likely because of increases in importation of ESBL-EK cases from the community. Further studies are needed on elucidating optimal infection control interventions to limit spread of ESBL-producing organisms in the hospital setting.

Risk factors for gastrointestinal tract colonization with extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli and Klebsiella species in hospitalized patients.

We describe the prevalence of and risk factors for colonization with extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli and Klebsiella species (ESBL-EK) in hospitalized patients. The prevalence of colonization with ESBL-EK was 2.6%. Colonization was associated with cirrhosis, longer duration of hospital stay prior to surveillance, and prior exposure to clindamycin or meropenem.

Prevalence of CTX-M beta-lactamases in Philadelphia, Pennsylvania.

CTX-M beta-lactamases were thought to be rare in the United States, but a recent study in Texas showed that up to 70% of extended-spectrum beta-lactamase (ESBL)-containing members of the Enterobacteriaceae family were CTX-M positive (J. S. Lewis, M. Herrera, B. Wickes, J. E. Patterson, and J. H. Jorgensen, Antimicrob. Agents Chemother. 51:4015-4021, 2007). We used PCR to detect CTX-M in all 291 extended-spectrum cephalosporin-resistant gram-negative bacteria isolated in our laboratory during 2007. Thirty (48%) Escherichia coli isolates, 6 (3%) Klebsiella sp. isolates, and 7 (100%) Proteus mirabilis isolates tested were CTX-M positive, with 15% of all Enterobacteriaceae tested being positive. The E. coli CTX-M groups were I (57%), IV (37%), II (3%), and not groupable (3%); three of the group IV isolates were positive for CTX-M-18, and three of the group I isolates were positive for CTX-M-15. One of seven positive P. mirabilis isolates was in group II, with the remainder being positive for a CTX-M-25-like beta-lactamase; and 33% of the Klebsiella sp. isolates were in group I or IV, with the remainder not being in groups I to IV. CTX-M-producing bacteria were isolated from urine (n = 13), blood (n = 13), wounds (n = 12), and the respiratory tract (n = 4). All 31 CTX-M-positive isolates tested for the presence of ESBL were confirmed to produce ESBLs by the use of tests recommended by the CLSI. Pulsed-field gel electrophoresis of the CTX-M-positive isolates showed that six P. mirabilis isolates were clonal and that there were seven different E. coli clusters. Five of seven P. mirabilis isolates were from blood cultures. The CLSI tests for the confirmation of ESBL production reliably detect these isolates if both cefotaxime and ceftazidime are tested, but only about half would be classified as a possible CTX-M producers on the basis of the antibiogram alone. A new panprimer set increases the ability to detect CTX-M-producing strains. CTX-M-positive bacteria are common in our geographic region, are often invasive, and, with the exception of P. mirabilis, are multiclonal.

Surveillance cultures for detection of methicillin-resistant Staphylococcus aureus: diagnostic yield of anatomic sites and comparison of provider- and patient-collected samples.

We studied provider- and patient-collected samples from multiple anatomic sites to determine the yield for detection of methicillin-resistant Staphylococcus aureus (MRSA). Sampling of multiple sites was required to achieve a sensitivity of more than 90% for MRSA colonization. Groin and perineum samples yielded positive results significantly more often for community-onset MRSA than for hospital-onset MRSA. Agreement rates between provider- and patient-collected swab specimens were excellent.

Legionella pneumophila NudA Is a Nudix hydrolase and virulence factor.

We studied the identity and function of the 528-bp gene immediately upstream of Legionella pneumophila F2310 ptsP (enzyme I(Ntr)). This gene, nudA, encoded for a Nudix hydrolase based on the inferred protein sequence. NudA had hydrolytic activity typical of other Nudix hydrolases, such as Escherichia coli YgdP, in that Ap(n)A's, in particular diadenosine pentaphosphate (Ap(5)A), were the preferred substrates. NudA hydrolyzed Ap(5)A to ATP plus ADP. Both ptsP and nudA were cotranscribed. Bacterial two-hybrid analysis showed no PtsP-NudA interactions. Gene nudA was present in 19 of 20 different L. pneumophila strains tested and in 5 of 10 different Legionella spp. other than L. pneumophila. An in-frame nudA mutation was made in L. pneumophila F2310 to determine the phenotype. The nudA mutant was an auxotroph that grew slowly in liquid and on solid media and had a smaller colony size than its parent. In addition, the mutant was more salt resistant than its parent and grew very poorly at 25 degrees C; all of these characteristics, as well as auxotrophy and slow-growth rate, were reversed by transcomplementation with nudA. The nudA mutant was outcompeted by about fourfold by the parent in competition studies in macrophages; transcomplementation almost completely restored this defect. Competition studies in guinea pigs with L. pneumophila pneumonia showed that the nudA mutant was outcompeted by its parent in both lung and spleen. NudA is of major importance for resisting stress in L. pneumophila and is a virulence factor.