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BackgroundThe CLEAR Trial demonstrated that a multisite body decolonization regimen reduced post-discharge infection and hospitalization in methicillin-resistant Staphylococcus aureus (MRSA) carriers. Here, we describe decolonization efficacy.MethodsWe performed a large, multicenter, randomized clinical trial of MRSA decolonization among adult patients after hospital discharge with MRSA infection or colonization. Participants were randomized 1:1 to either MRSA prevention education or education plus decolonization with topical chlorhexidine, oral chlorhexidine, and nasal mupirocin. Participants were swabbed in the nares, throat, axilla/groin, and wound (if applicable) at baseline and 1, 3, 6, and 9 months after randomization. The primary outcomes of this study are follow-up colonization differences between groups.ResultsAmong 2121 participants, 1058 were randomized to decolonization. By 1 month, MRSA colonization was lower in the decolonization group compared with the education-only group (odds ration [OR] = 0.44; 95% confidence interval [CI], .36-.54; P ≤ .001). A similar magnitude of reduction was seen in the nares (OR = 0.34; 95% CI, .27-.42; P < .001), throat (OR = 0.55; 95% CI, .42-.73; P < .001), and axilla/groin (OR = 0.57; 95% CI, .43-.75; P < .001). These differences persisted through month 9 except at the wound site, which had a relatively small sample size. Higher regimen adherence was associated with lower MRSA colonization (P ≤ .01).ConclusionsIn a randomized, clinical trial, a repeated post-discharge decolonization regimen for MRSA carriers reduced MRSA colonization overall and at multiple body sites. Higher treatment adherence was associated with greater reductions in MRSA colonization.

In a prospective cohort study, we compared a 2-swabs-per-nostril 5% iodophor regimen with a 1-swab-per-nostril 10% iodophor regimen on methicillin-resistant Staphylococcus aureus carriage in nursing-home residents. Compared with baseline, both single-swab and double-swab regimens resulted in an identical 40% reduction in nasal carriage and 60% reduction in any carriage, skin or nasal.

BackgroundCarbapenem-resistant Enterobacterales (CRE) are an urgent global health threat. Inferring the dynamics of local CRE dissemination is currently limited by our inability to confidently trace the spread of resistance determinants to unrelated bacterial hosts. Whole-genome sequence comparison is useful for identifying CRE clonal transmission and outbreaks, but high-frequency horizontal gene transfer (HGT) of carbapenem resistance genes and subsequent genome rearrangement complicate tracing the local persistence and mobilization of these genes across organisms.MethodsTo overcome this limitation, we developed a new approach to identify recent HGT of large, near-identical plasmid segments across species boundaries, which also allowed us to overcome technical challenges with genome assembly. We applied this to complete and near-complete genome assemblies to examine the local spread of CRE in a systematic, prospective collection of all CRE, as well as time- and species-matched carbapenem-susceptible Enterobacterales, isolated from patients from four US hospitals over nearly 5 years.ResultsOur CRE collection comprised a diverse range of species, lineages, and carbapenem resistance mechanisms, many of which were encoded on a variety of promiscuous plasmid types. We found and quantified rearrangement, persistence, and repeated transfer of plasmid segments, including those harboring carbapenemases, between organisms over multiple years. Some plasmid segments were found to be strongly associated with specific locales, thus representing geographic signatures that make it possible to trace recent and localized HGT events. Functional analysis of these signatures revealed genes commonly found in plasmids of nosocomial pathogens, such as functions required for plasmid retention and spread, as well survival against a variety of antibiotic and antiseptics common to the hospital environment.ConclusionsCollectively, the framework we developed provides a clearer, high-resolution picture of the epidemiology of antibiotic resistance importation, spread, and persistence in patients and healthcare networks.

PurposeTo report a case of orbital coccidiomycosis in an otherwise healthy 11-month-old male.ObservationsAn 11-month-old male presented to his pediatrician with parental complaints of swelling, erythema, and pain of the right orbit that increased over ten days in the absence of constitutional symptoms. The child's parents reported an earlier fall onto a carpeted floor. After four weeks of conservative treatment and a course of oral cephalexin, he developed a fever, increased erythema, and palpable enlargement of a mass posterior to the lower eyelid. Ultrasound revealed an encysted mass in the inferior orbit, suggestive of an abscess. Urgent ophthalmic referral led to incision and drainage via orbitotomy. Culture, histopathology, and serological testing were positive for Coccidioides spp.. Blood studies revealed mild anemia and thrombocytosis. There was complete resolution of symptoms after surgical drainage and several weeks of oral fluconazole.Conclusion and importanceWe describe a patient with orbital coccidiomycosis without apparent systemic involvement, following what was most likely an unrelated minor trauma. Despite being rare, orbital coccidiomycosis should be considered as a primary manifestation of infection when ocular inflammation is encountered, especially in endemic regions.

ObjectiveDetermine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus spp. (VRE), extended-spectrum beta-lactamase producing organisms (ESBLs), and carbapenem-resistant Enterobacteriaceae (CRE) among residents and in the environment of nursing homes (NHs).DesignPoint prevalence sampling of residents and environmental sampling of high-touch objects in resident rooms and common areas.SettingTwenty-eight NHs in Southern California from 2016 to 2017.ParticipantsNH participants in Project PROTECT, a cluster-randomized trial of enhanced bathing and decolonization vs routine care.MethodsFifty residents were randomly sampled per NH. Twenty objects were sampled, including 5 common room objects plus 5 objects in each of 3 rooms (ambulatory, total care, and dementia care residents).ResultsA total of 2797 swabs were obtained from 1400 residents in 28 NHs. Median prevalence of multidrug-resistant organism (MDRO) carriage per NH was 50% (range: 24%-70%). Median prevalence of specific MDROs were as follows: MRSA, 36% (range: 20%-54%); ESBL, 16% (range: 2%-34%); VRE, 5% (range: 0%-30%); and CRE, 0% (range: 0%-8%). A median of 45% of residents (range: 24%-67%) harbored an MDRO without a known MDRO history. Environmental MDRO contamination was found in 74% of resident rooms and 93% of common areas.Conclusions and implicationsIn more than half of the NHs, more than 50% of residents were colonized with MDROs of clinical and public health significance, most commonly MRSA and ESBL. Additionally, the vast majority of resident rooms and common areas were MDRO contaminated. The unknown submerged portion of the iceberg of MDRO carriers in NHs may warrant changes to infection prevention and control practices, particularly high-fidelity adoption of universal strategies such as hand hygiene, environmental cleaning, and decolonization.

Background: Addressing the high burden of multidrug-resistant organisms (MDROs) in nursing homes is a public health priority. High interfacility transmission may be attributed to inadequate infection prevention practices, shared living spaces, and frequent care needs. We assessed the contribution of roommates to the likelihood of MDRO carriage in nursing homes. Methods: We performed a secondary analysis of the SHIELD OC (Shared Healthcare Intervention to Eliminate Life-threatening Dissemination of MDROs in Orange County, CA) Project, a CDC-funded regional decolonization intervention to reduce MDROs among 38 regional facilities (18 nursing homes, 3 long-term acute-care hospitals, and 17 hospitals). Decolonization in participating nursing homes involved routine chlorhexidine bathing plus nasal iodophor (Monday through Friday, twice daily every other week) from April 2017 through July 2019. MDRO point-prevalence assessments involving all residents at 16 nursing homes conducted at the end of the intervention period were used to determine whether having a roommate was associated with MDRO carriage. Nares, bilateral axilla/groin, and perirectal swabs were processed for methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococcus (VRE), extended-spectrum β-lactamase (ESBL)–producing Enterobacteriaceae, and carbapenem-resistant Enterobacteriaceae (CRE). Generalized linear mixed models assessed the impact of maximum room occupancy on MDRO prevalence when clustering by room and hallway, and adjusting for the following factors: nursing home facility, age, gender, length-of-stay at time of swabbing, bedbound status, known MDRO history, and presence of urinary or gastrointestinal devices. CRE models were not run due to low counts. Results: During the intervention phase, 1,451 residents were sampled across 16 nursing homes. Overall MDRO prevalence was 49%. In multivariable models, we detected a significant increasing association of maximum room occupants and MDRO carriage for MRSA but not other MDROs. For MRSA, the adjusted odds ratios for quadruple-, triple-, and double-occupancy rooms were 3.5, 3.6, and 2.8, respectively, compared to residents in single rooms (P = .013). For VRE, these adjusted odds ratios were 0.3, 0.3, and 0.4, respectively, compared to residents in single rooms (P = NS). For ESBL, the adjusted odds ratios were 0.9, 1.1, and 1.5, respectively, compared to residents in single rooms (P = nonsignificant). Conclusions: Nursing home residents in shared rooms were more likely to harbor MRSA, suggesting MRSA transmission between roommates. Although decolonization was previously shown to reduce MDRO prevalence by 22% in SHIELD nursing homes, this strategy did not appear to prevent all MRSA transmission between roommates. Additional efforts involving high adherence hand hygiene, environmental cleaning, and judicious use of contact precautions are likely needed to reduce transmission between roommates in nursing homes. Funding: None Disclosures: Gabrielle M. Gussin, Stryker (Sage Products): Conducting studies in which contributed antiseptic product is provided to participating hospitals and nursing homes. Clorox: Conducting studies in which contributed antiseptic product is provided to participating hospitals and nursing homes. Medline: Conducting studies in which contributed antiseptic product is provided to participating hospitals and nursing homes. Xttrium: Conducting studies in which contributed antiseptic product is provided to participating hospitals and nursing homes.

Distinguished Oral Background: Shared Healthcare Intervention to Eliminate Life-threatening Dissemination of MDROs in Orange County, California (SHIELD OC) was a CDC-funded regional decolonization intervention from April 2017 through July 2019 involving 38 hospitals, nursing homes (NHs), and long-term acute-care hospitals (LTACHs) to reduce MDROs. Decolonization in NH and LTACHs consisted of universal antiseptic bathing with chlorhexidine (CHG) for routine bathing and showering plus nasal iodophor decolonization (Monday through Friday, twice daily every other week). Hospitals used universal CHG in ICUs and provided daily CHG and nasal iodophor to patients in contact precautions. We sought to evaluate whether decolonization reduced hospitalization and associated healthcare costs due to infections among residents of NHs participating in SHIELD compared to nonparticipating NHs. Methods: Medicaid insurer data covering NH residents in Orange County were used to calculate hospitalization rates due to a primary diagnosis of infection (counts per member quarter), hospital bed days/member-quarter, and expenditures/member quarter from the fourth quarter of 2015 to the second quarter of 2019. We used a time-series design and a segmented regression analysis to evaluate changes attributable to the SHIELD OC intervention among participating and nonparticipating NHs. Results: Across the SHIELD OC intervention period, intervention NHs experienced a 44% decrease in hospitalization rates, a 43% decrease in hospital bed days, and a 53% decrease in Medicaid expenditures when comparing the last quarter of the intervention to the baseline period (Fig. 1). These data translated to a significant downward slope, with a reduction of 4% per quarter in hospital admissions due to infection (P < .001), a reduction of 7% per quarter in hospitalization days due to infection (P < .001), and a reduction of 9% per quarter in Medicaid expenditures (P = .019) per NH resident. Conclusions: The universal CHG bathing and nasal decolonization intervention adopted by NHs in the SHIELD OC collaborative resulted in large, meaningful reductions in hospitalization events, hospitalization days, and healthcare expenditures among Medicaid-insured NH residents. The findings led CalOptima, the Medicaid provider in Orange County, California, to launch an NH incentive program that provides dedicated training and covers the cost of CHG and nasal iodophor for OC NHs that enroll. Funding: None Disclosures: Gabrielle M. Gussin, University of California, Irvine, Stryker (Sage Products): Conducting studies in which contributed antiseptic product is provided to participating hospitals and nursing homes. Clorox: Conducting studies in which contributed antiseptic product is provided to participating hospitals and nursing homes. Medline: Conducting studies in which contributed antiseptic product is provided to participating hospitals and nursing homes. Xttrium: Conducting studies in which contributed antiseptic product is provided to participating hospitals and nursing homes.

Background: More than half of nursing home (NH) residents harbor a multidrug-resistant organism (MDRO), and MDRO contamination of the environment is common. Whether NH decolonization of residents reduces MDRO contamination remains unclear. The PROTECT trial was a cluster-randomized trial of decolonization versus routine care in 28 California NHs from April 2017 through December 2018. Decolonization involved chlorhexidine bathing plus nasal iodophor (Monday–Friday, every other week), and it reduced resident nares and skin MDRO colonization by 36%. Methods: We swabbed high-touch objects in resident rooms and common areas for MDROs before and after the 3-month decolonization phase-in (April–July 2017). Five high-touch objects (bedrail, call button and TV remote, doorknob, light switch, and bathroom handles) were swabbed in 3 resident rooms per NH based on care needs (Alzheimer’s disease and related dementias (ADRD), ie, total care; ADRD, ambulatory care; and short stay). Five high-touch objects were also swabbed in the common area (nursing station, table, chair, railing, and drinking fountain). Swabs were processed for methicillin-resistant S. aureus (MRSA), vancomycin-resistant Enterococcus (VRE), extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae, and carbapenem-resistant Enterobacteriaceae (CRE). We used generalized linear mixed models to assess the impact of decolonization on MDRO environmental contamination when clustering by NH and room and adjusting for room type and object because unclustered and unadjusted results are likely to be inaccurate. Results: A high proportion of rooms were contaminated with any MDRO in control NHs: 43 of 56 (77%) in the baseline period and 46 of 56 (82%) in the intervention period. In contrast, decolonization NHs had similar baseline contamination (45 of 56, 80%) but lower intervention MDRO contamination (29 of 48, 60%). When evaluating the intervention impact using multivariable models, decolonization was associated with significantly less room contamination for any MDRO (OR, 0.25; 95% CI, 0.06–0.96; P = .04) and MRSA (OR, 0.16; 95% CI, 0.05–0.55; P = .004) but nonsignificant reductions in VRE contamination (OR, 0.86; 95% CI, 0.23–3.13) and ESBL contamination (OR, 0.13; 95% CI, 0.01–1.62). CRE was not modeled due to rare counts (2 rooms total). In addition, room type was important, with common areas associated with 5-fold, 9-fold, and 3-fold higher contamination with any MDRO, MRSA, and VRE, respectively, compared with short-stay rooms. Conclusions: The high burden of MDROs in NHs calls for universal prevention strategies that can protect all residents. Although decolonization was associated with an 84% reduction in odds of MRSA contamination of inanimate room objects, significant reductions in VRE or ESBL contamination were not seen, possibly due to the lower proportion of baseline contamination due to these organisms. Multimodal strategies are needed to address high levels of MDRO contamination in NHs.Funding: NoneDisclosures: Gabrielle Gussin: Stryker (Sage Products): Conducting studies in which contributed antiseptic product is provided to participating hospitals and nursing homes. Clorox: Conducting studies in which contributed antiseptic product is provided to participating hospitals and nursing homes. Medline: Conducting studies in which contributed antiseptic product is provided to participating hospitals and nursing homes. Xttrium: Conducting studies in which contributed antiseptic product is provided to participating hospitals and nursing homes.

Sepsis due to antimicrobial resistant pathogens is a major health problem worldwide. The inability to rapidly detect and thus treat bacteria with appropriate agents in the early stages of infections leads to excess morbidity, mortality, and healthcare costs. Here we report a rapid diagnostic platform that integrates a novel one-step blood droplet digital PCR assay and a high throughput 3D particle counter system with potential to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood specimens, without requiring culture and sample processing steps. Using CTX-M-9 family ESBLs as a model system, we demonstrated that our technology can simultaneously achieve unprecedented high sensitivity (10 CFU per ml) and rapid sample-to-answer assay time (one hour). In head-to-head studies, by contrast, real time PCR and BioRad ddPCR only exhibited a limit of detection of 1000 CFU per ml and 50-100 CFU per ml, respectively. In a blinded test inoculating clinical isolates into whole blood, we demonstrated 100% sensitivity and specificity in identifying pathogens carrying a particular resistance gene. We further demonstrated that our technology can be broadly applicable for targeted detection of a wide range of antibiotic resistant genes found in both Gram-positive (vanA, nuc, and mecA) and Gram-negative bacteria, including ESBLs (blaCTX-M-1 and blaCTX-M-2 families) and CREs (blaOXA-48 and blaKPC), as well as bacterial speciation (E. coli and Klebsiella spp.) and pan-bacterial detection, without requiring blood culture or sample processing. Our rapid diagnostic technology holds great potential in directing early, appropriate therapy and improved antibiotic stewardship in combating bloodstream infections and antibiotic resistance.

As medical schools seek to address the growing disparity between the socioeconomic makeup of their students and the general population, it is important to understand the academic trajectory of disadvantaged students. We used a locally-developed multicomponent socioeconomic disadvantage (SED) measure and the self-designated disadvantaged (SDA) question ["yes" (+) or "no" (-)] from the American Medical College Application Service application to examine academic performance of students from three disadvantaged categories (high SED/SDA+, high SED/SDA-, and low SED/SDA+); with low SED/SDA-as the reference group across five California schools. Compared with reference, the DA+ subgroups scored lower on USMLE Step 1 and Step 2 Clinical Knowledge examinations and received fewer clerkship Honors. After adjustment for academic metrics and sociodemographic variables, high SED subgroups performed similarly to reference, but performance gaps for low SED/SDA+ students persisted. Medical schools must better understand the institutional and other drivers of academic success in disadvantaged students.

BackgroundMultidrug-resistant organisms (MDROs) spread between hospitals, nursing homes (NHs), and long-term acute care facilities (LTACs) via patient transfers. The Shared Healthcare Intervention to Eliminate Life-threatening Dissemination of MDROs in Orange County is a regional public health collaborative involving decolonization at 38 healthcare facilities selected based on their high degree of patient sharing. We report baseline MDRO prevalence in 21 NHs/LTACs.MethodsA random sample of 50 adults for 21 NHs/LTACs (18 NHs, 3 LTACs) were screened for methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus spp. (VRE), extended-spectrum β-lactamase-producing organisms (ESBL), and carbapenem-resistant Enterobacteriaceae (CRE) using nares, skin (axilla/groin), and peri-rectal swabs. Facility and resident characteristics associated with MDRO carriage were assessed using multivariable models clustering by person and facility.ResultsPrevalence of MDROs was 65% in NHs and 80% in LTACs. The most common MDROs in NHs were MRSA (42%) and ESBL (34%); in LTACs they were VRE (55%) and ESBL (38%). CRE prevalence was higher in facilities that manage ventilated LTAC patients and NH residents (8% vs

PurposeTo compare the predictive validities of medical school admissions multiple mini-interviews (MMIs) and traditional interviews (TIs).MethodThis longitudinal observational study of 2011-2013 matriculants to five California public medical schools examined the associations of MMI scores (two schools) and TI scores (three schools) with subsequent academic performance. Regression models adjusted for sociodemographics and undergraduate academic metrics examined associations of standardized mean MMI and TI scores with United States Medical Licensing Examination Step 1 and Step 2 Clinical Knowledge (CK) scores and, for required clerkships, with mean National Board of Medical Examiners Clinical Science subject (shelf) exam score and number of honors grades.ResultsOf the 1,460 medical students, 746 (51.1%) interviewed at more than one study school; 579 (39.7%) completed at least one MMI and at least one TI. Neither interview type was associated with Step 1 scores. Higher MMI scores were associated with more clerkship honors grades (adjusted incidence rate ratio [AIRR] 1.28 more [95% CI 1.18, 1.39; P < .01] per SD increase) and higher shelf exam and Step 2 CK scores (adjusted mean 0.73 points higher [95% CI 0.28, 1.18; P < .01] and 1.25 points higher [95% CI 0.09, 2.41; P = .035], respectively, per SD increase). Higher TI scores were associated only with more honors grades (AIRR 1.11 more [95% CI 1.01, 1.20; P = .03] per SD increase).ConclusionsMMI scores were more strongly associated with subsequent academic performance measures than were TI scores.

Autophagy can either antagonize or promote intracellular bacterial growth, depending on the pathogen. Here, we investigated the role of autophagy during a pulmonary infection with the obligate intracellular pathogen, Chlamydia pneumoniae (CP). In mouse embryonic fibroblasts (MEFs) or macrophages, deficiency of autophagy pathway components led to enhanced CP replication, suggesting that autophagy exerts a bactericidal role. However, in vivo, mice with myeloid-specific deletion of the autophagic protein ATG16L1 suffered increased mortality during CP infection, neutrophilia, and increased inflammasome activation despite no change in bacterial burden. Induction of autophagy led to reduced CP replication in vitro, but impaired survival in CP-infected mice, associated with an initial reduction in IL-1β production, followed by enhanced neutrophil recruitment, defective CP clearance, and later inflammasome activation and IL-1β production, which drove the resulting mortality. Taken together, our data suggest that a delicate interplay exists between autophagy and inflammasome activation in determining the outcome of CP infection, perturbation of which can result in inflammatory pathology or unrestricted bacterial growth.

PURPOSE:In single-school studies, multiple mini-interview (MMI) and traditional interview (TI) scores are associated with acceptance offers. Unexamined is whether scores at one school are associated with acceptance at other schools; such analyses would mitigate single-school design biases and better estimate how well interviews capture desired applicant attributes. Using data from the 5 California Longitudinal Evaluation of Admissions Practices (CA-LEAP) medical schools, the authors examined associations of MMI and TI scores with acceptance offers within and across schools. METHOD:The analyses included applicants who interviewed at ≥1 CA-LEAP school during the 2011-2013 admissions cycles. Three CA-LEAP schools employed TIs and 2 employed MMIs. Interview scores were standardized (z scores: mean = 0, SD = 1), and associations with acceptance offers were examined within and across schools in analyses stratified by school, adjusting for applicant sociodemographics, academic metrics, year, and total number of interviews. RESULTS:Of 4,993 applicants interviewed, 428 (8.6%) interviewed at both MMI schools, 681 (13.6%) at ≥2 TI schools, and 1,327 (26.6%) at ≥1 MMI and ≥1 TI school. For each school, acceptance was associated with interview score at that school and also with interview scores at the other 4 schools. Cross-school associations of MMI versus TI scores with acceptance did not differ statistically. CONCLUSIONS:Interview score at a given school was associated with acceptance at the other 4 schools, with no significant differences in associations for MMIs versus TIs. The findings suggest both MMIs and TIs captured attributes valued by admissions teams across CA-LEAP schools.

BackgroundMany medical schools use admissions Multiple Mini-Interviews (MMIs) rather than traditional interviews (TIs), partly because MMIs are thought to be more reliable. Yet prior studies examined single-school samples of candidates completing either an MMI or TI (not both). Using data from five California public medical schools, the authors examined the within- and between-school reliabilities of TIs and MMIs.MethodsThe analyses included applicants interviewing at ≥1 of the five schools during 2011-2013. Three schools employed TIs (TI1, TI2, TI3) and two employed MMIs (MMI1, MMI2). Mixed linear models accounting for nesting of observations within applicants examined standardized TI and MMI scores (mean = 0, SD = 1), adjusting for applicant socio-demographics, academic metrics, year, number of interviews, and interview date.ResultsA total of 4993 individuals (completing 7516 interviews [TI = 4137, MMI = 3379]) interviewed at ≥1 school; 428 (14.5%) interviewed at both MMI schools and 687 (20.2%) at more than one TI school. Within schools, inter-interviewer consistency was generally qualitatively lower for TI1, TI2, and TI3 (Pearson's r 0.07, 0.13, and 0.29, and Cronbach's α, 0.40, 0.44, and 0.61, respectively) than for MMI1 and MMI 2 (Cronbach's α 0.68 and 0.60, respectively). Between schools, the adjusted intraclass correlation coefficient was 0.27 (95% CI 0.20-0.35) for TIs and 0.47 (95% CI 0.41-0.54) for MMIs.ConclusionsWithin and between-school reliability was qualitatively higher for MMIs than for TIs. Nonetheless, TI reliabilities were higher than anticipated from prior literature, suggesting TIs may not need to be abandoned on reliability grounds if other factors favor their use.

Abstract Background MDROs can spread between hospitals, nursing homes (NH), and long-term acute care facilities (LTACs) via shared patients. SHIELD OC is a regional decolonization collaborative involving 38 of 104 countywide adult facilities identified by their high degree of direct and indirect patient sharing with one another. We report baseline MDRO prevalence in these facilities. Methods Adult patients in 38 facilities (17 hospitals, 18 NHs, 3 LTACs) underwent point-prevalence screening between September 2016–April 2017 for MRSA, VRE, ESBL, and CRE using nares, skin (axilla/groin), and peri-rectal swabs. In NHs and LTACs, residents were randomly selected until 50 sets of swabs were obtained. Swabbing in hospitals involved all patients in contact precautions. An additional set of swabs were also performed for all LTAC admissions from November 2016–February 2017. Results The overall prevalence of any MDRO among patients was 64% (44%–88%) in NHs, 80% (range 72%–86%) in LTACs, and 64% (54–84%) in hospitals (contact precaution patients) (Table 1). Only 25%, 64%, and 81% of patients were already known to harbor an MDRO in NHs, LTACs, and hospitals, respectively. Known MDRO patients also harbored another MDRO 49%, 63%, and 34% of the time for NHs, LTACs, and hospitals, respectively. In LTACs, MDRO point prevalence was 38% higher than the usual admission prevalence (65% higher for MRSA, 34% higher for VRE, 95% higher for ESBL, and 50% higher for CRE). Conclusion MDRO carriage in highly inter-connected NHs and LTACs was widespread, rivaling that found in hospitalized patients on contact precautions. MRSA, VRE, and ESBL carriage far outnumbered CRE carriage. A history of MDRO was insensitive for identifying MDRO carriers, and many patients carried multiple MDROs. The extensive MDRO burden and transmission in long-term care settings suggests that regional MDRO prevention efforts must include MDRO control in long-term care facilities. Disclosures R. D. Singh, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; 3M: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium Laboratories: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; J. A. McKinnell, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; 3M: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; L. G. Miller, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; 3M: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; K. Kleinman, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium Laboratories: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Molnlycke: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; 3M: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; L. Heim, Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; 3M: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; T. D. Dutciuc, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; 3M: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; M. Estevez, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; 3M: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; G. Gussin, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium Laboratories: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; L’Oreal: Consultant, Consulting fee; J. Chang, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium Laboratories: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; 3M: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; E. M. Peterson, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium Laboratories: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; B. Y. Lee, GSK: Consultant, Consulting fee; R. A. Weinstein, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Molnlycke: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; OpGen Company: Study support, Provided services at no charge; M. K. Hayden, Sage Products: Receipt of contributed product, Sage is contributing product to healthcare facilities participating in a regional collaborative on which I am a co-investigator. Neither I nor my hospital receive product.; Clorox: Receipt of contributed product, Research support; CDC: Grant Investigator and Receipt of contributed product, Research grant; Molnlycke: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; OpGen Company: Study support, Provided services at no charge for studies; S. K. Gohil, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium Laboratories: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; S. Park, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium Laboratories: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; S. Tam, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; 3M: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium Laboratories: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; R. Saavedra, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium Laboratories: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; S. Yamaguchi, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium Laboratories: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; H. Custodio, Xttrium Laboratories: Study coordination, Conducting studies in healthcare facilities that are receiving contributed product; Sage Products: Study coordination, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Study coordination, Conducting studies in healthcare facilities that are receiving contributed product; J. Nguyen, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium Laboratories: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; T. Tjoa, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium Laboratories: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; 3M: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; J. He, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium Laboratories: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; 3M: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; M. H. Coady, Sage Products: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Molnlycke: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Xttrium Laboratories: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; Clorox: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; R. Platt, Sage Products: Receipt of contributed product, Conducting clinical studies in which participating healthcare facilities are receiving contributed product; Xttrium Laboratories: Receipt of contributed product, Conducting clinical studies in which participating healthcare facilities are receiving contributed product; Clorox: Receipt of contributed product, Conducting clinical studies in which participating healthcare facilities are receiving contributed product; receive research funds from Clorox, but Clorox has no role in the design; Molnlycke: Receipt of contributed product, Conducting studies in healthcare facilities that are receiving contributed product; S. S. Huang, Sage Products: Receipt of contributed product, Conducting studies in which participating healthcare facilities are receiving contributed product (no contribution in submitted abstract), Participating healthcare facilities in my studies received contributed product; Xttrium Laboratories: Receipt of contributed product, Conducting studies in which participating healthcare facilities are receiving contributed product (no contribution in submitted abstract), Participating healthcare facilities in my studies received contributed product; Clorox: Receipt of contributed product, Conducting studies in which participating healthcare facilities are receiving contributed product (no contribution in submitted abstract), Participating healthcare facilities in my studies received contributed product; 3M: Receipt of contributed product, Conducting studies in which participating healthcare facilities are receiving contributed product (no contribution in submitted abstract), Participating healthcare facilities in my studies received contributed product; Molnlycke: Receipt of contributed product, Conducting studies in which participating healthcare facilities are receiving contributed product (no contribution in submitted abstract), Participating healthcare facilities in my studies received contributed product

Abstract Background The majority of healthcare-associated infections due to MDROs occur in the post-discharge setting. Understanding MDRO spread and containment in NHs can help identify infection prevention activities needed to care for vulnerable patients in a medical home setting. Methods We conducted a baseline point prevalence study of MDRO colonization in residents of 28 Southern California NHs participating in a decolonization trial. In Fall 2016, residents were randomly sampled to obtain a set of 50 nares and skin (axilla/groin) swabs from each NH. Nasal swabs were processed for MRSA and skin swabs were processed for MRSA, VRE, ESBL, and CRE. In addition, environmental swabs were collected from high touch objects in resident rooms (bedrail, call button/TV remote, door knobs, light switch, bathroom) and common areas (nursing station, table, chair, railing, and drinking fountain). Results A total of 2,797 body swabs were obtained from 1400 residents. Overall, 48.6% (N = 680) of residents harbored MDROs. MRSA was found in 37% of residents (29.5% nares, 24.4% skin), followed by ESBL in 16% (Table 1). Resident MDRO status was only known for 11% of MRSA (59/518), 18% ESBL (40/228), 4% VRE (4/99), and none of the CRE (0/13) carriers. Colonization did not differ between long stay (48.8%, 534/1094) vs. post-acute (47.7%, 146/306) residents (P = NS), but bedbound residents were more likely to be MDRO colonized (58.7%, 182/310) vs. ambulatory residents (45.7%, 497/1088, P

Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to the antibiotic era. Multiple different species can exhibit resistance due to many different mechanisms, and many different mobile elements are capable of transferring resistance between lineages. We prospectively sampled CRE from hospitalized patients from three Boston-area hospitals, together with a collection of CRE from a single California hospital, to define the frequency and characteristics of outbreaks and determine whether there is evidence for transfer of strains within and between hospitals and the frequency with which resistance is transferred between lineages or species. We found eight species exhibiting resistance, with the majority of our sample being the sequence type 258 (ST258) lineage of Klebsiella pneumoniae There was very little evidence of extensive hospital outbreaks, but a great deal of variation in resistance mechanisms and the genomic backgrounds carrying these mechanisms. Local transmission was evident in clear phylogeographic structure between the samples from the two coasts. The most common resistance mechanisms were KPC (K. pneumoniae carbapenemases) beta-lactamases encoded by blaKPC2, blaKPC3, and blaKPC4, which were transferred between strains and species by seven distinct subgroups of the Tn4401 element. We also found evidence for previously unrecognized resistance mechanisms that produced resistance when transformed into a susceptible genomic background. The extensive variation, together with evidence of transmission beyond limited clonal outbreaks, points to multiple unsampled transmission chains throughout the continuum of care, including asymptomatic carriage and transmission of CRE. This finding suggests that to control this threat, we need an aggressive approach to surveillance and isolation.

Nursing home residents are at risk for acquiring and transmitting MDROs. A serial point-prevalence study of 605 residents in 3 facilities using random sampling found MDRO colonization in 45% of residents: methicillin-resistant Staphylococcus aureus (MRSA, 26%); extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL, 17%); vancomycin-resistant Enterococcus spp. (VRE, 16%); carbapenem-resistant Enterobacteriaceae (CRE, 1%). MDRO colonization was associated with history of MDRO, care needs, incontinence, and catheters. Infect Control Hosp Epidemiol 2016;1485-1488.

UnlabelledThe Scc4 protein (CT663) of the pathogenic bacterium Chlamydia has been described as a type III secretion (T3S) chaperone as well as an inhibitor of RNA polymerase. To examine if these roles are connected, we first investigated physical interactions between Chlamydia trachomatis Scc4 and the T3S chaperone Scc1 and a T3S substrate, CopN. In a yeast 3-hybrid assay, Scc4, Scc1, and CopN were all required to detect an interaction, which suggests that these proteins form a trimolecular complex. We also detected interactions between any two of these three T3S proteins in a pulldown assay using only recombinant proteins. We next determined whether these interactions affected the function of Scc4 as an inhibitor of RNA transcription. Using Escherichia coli as a heterologous in vivo system, we demonstrated that expression of C. trachomatis Scc4 led to a drastic decrease in transcript levels for multiple genes. However, coexpression of Scc4 with Scc1, CopN, or both alleviated Scc4-mediated inhibition of transcription. Scc4 expression also severely impaired E. coli growth, but this growth defect was reversed by coexpression of Scc4 with Scc1, CopN, or both, suggesting that the inhibitory effect of Scc4 on transcription and growth can be antagonized by interactions between Scc4, Scc1, and CopN. These findings suggest that the dual functions of Scc4 may serve as a bridge to link T3S and the regulation of gene expression in Chlamydia.ImportanceThis study investigates a novel mechanism for regulating gene expression in the pathogenic bacterium Chlamydia. The Chlamydia type III secretion (T3S) chaperone Scc4 has been shown to inhibit transcription by RNA polymerase. This study describes physical interactions between Scc4 and the T3S proteins Scc1 and CopN. Furthermore, Chlamydia Scc1 and CopN antagonized the inhibitory effects of Scc4 on transcription and growth in a heterologous Escherichia coli system. These results provide evidence that transcription in Chlamydia can be regulated by the T3S system through interactions between T3S proteins.

ObjectiveTo evaluate whether an ecologic inverse association exists between methicillin-susceptible Staphylococcus aureus (MSSA) prevalence and methicillin-resistant S. aureus (MRSA) prevalence in nursing homes.MethodsWe conducted a secondary analysis of a prospective cross-sectional study of S. aureus prevalence in 26 nursing homes across Orange County, California, from 2008-2011. Admission prevalence was assessed using bilateral nares swabs collected from all new residents within 3 days of admission until 100 swabs were obtained. Point prevalence was assessed from a representative sample of 100 residents. Swab samples were plated on 5% sheep blood agar and Spectra MRSA chromogenic agar. If MRSA was detected, no further tests were performed. If MRSA was not detected, blood agar was evaluated for MSSA growth. We evaluated the association between MRSA and MSSA admission and point prevalence using correlation and linear regression testing.ResultsWe collected 3,806 total swabs. MRSA and MSSA admission prevalence were not correlated (r = -0.40, P = .09). However, MRSA and MSSA point prevalence were negatively correlated regardless of whether MSSA prevalence was measured among all residents sampled (r = -0.67, P = .0002) or among those who did not harbor MRSA (r = -0.41, P = .04). This effect persisted in regression models adjusted for the percentage of residents with diabetes (β = -0.73, P = .04), skin lesions (β = -1.17, P = .002), or invasive devices (β = -1.4, P = .0006).ConclusionsThe inverse association between MRSA and MSSA point prevalence and minimal association on admission prevalence suggest MSSA carriage may protect against MRSA acquisition in nursing homes. The minimal association on admission prevalence further suggests competition may occur during nursing home stays.

The salicylidene acylhydrazide (SA) compounds have exhibited promising microbicidal properties. Previous reports have shown the SA compounds, using cell cultures, to exhibit activity against Chlamydia trachomatis, herpes simplex virus and HIV-1. In addition, using an animal model of a vaginal infection the SA compound INP0341, when dissolved in a liquid, was able to significantly protect mice from a vaginal infection with C. trachomatis. To expand upon this finding, in this report INP0341 was formulated as a vaginal gel, suitable for use in humans. Gelling agents (polymers) with inherent antimicrobial properties were chosen to maximize the total antimicrobial effect of the gel. In vitro formulation work generated a gel with suitable rheology and sustained drug release. A formulation containing 1 mM INP0341, 1.6 wt% Cremophor ELP (solubility enhancer) and 1.5 wt% poly(acrylic acid) (gelling and antimicrobial agent), was chosen for studies of efficacy and toxicity using a mouse model of a vaginal infection. The gel formulation was able to attenuate a vaginal challenge with C. trachomatis, serovar D. Formulations with and without INP0341 afforded protection, but the inclusion of INP0341 increased the protection. Mouse vaginal tissue treated with the formulation showed no indication of gel toxicity. The lack of toxicity was confirmed by in vitro assays using EpiVaginal tissues, which showed that a 24 h exposure to the gel formulation did not decrease the cell viability or the barrier function of the tissue. Therefore, the gel formulation described here appears to be a promising vaginal microbicide to prevent a C. trachomatis infection with the potential to be expanded to other sexually transmitted diseases.

Chlamydophila pneumoniae (CPn) is a common respiratory pathogen that causes a chronic and persistent airway infection. The elderly display an increased susceptibility and severity to this infection. However, the underlying mechanisms are not well understood. Dendritic cells (DCs) are the initiators and regulators of immune responses. Therefore, we investigated the role of DCs in the age-associated increased CPn infection in vitro in humans. Though the expression of activation markers was comparable between the two age groups, DCs from aged subjects secreted enhanced levels of proinflammatory mediators such as TNF-α and CXCL-10 in response to CPn. In contrast, the secretion of IL-10 and innate interferons, IFN-α and IFN-λ, was severely impaired in DCs from aged donors. The increased activation of DCs from aged subjects to CPn also resulted in enhanced proliferation of CD4 and CD8 T cells in a DC-T coculture. Furthermore, T cells primed with CPn-stimulated DCs from aged subjects secreted increased levels of IFN-γ and reduced levels of IL-10 compared to DCs obtained from young subjects. In summary, DCs from the elderly displayed enhanced inflammatory response to CPn which may result in airway remodeling and increase the susceptibility of the elderly to respiratory diseases such as asthma.

Chlorhexidine and mupirocin are used in health care facilities to eradicate methicillin-resistant Staphylococcus aureus (MRSA) carriage. The objective of this study was to assess the frequency of chlorhexidine and mupirocin resistance in isolates from nares carriers in multiple nursing homes and to examine characteristics associated with resistance. Nasal swab samples were collected from approximately 100 new admissions and 100 current residents in 26 nursing homes in Orange County, CA, from October 2008 to May 2011. MRSA isolates were tested for susceptibility by using broth microdilution, disk diffusion, and Etest; for genetic relatedness using pulsed-field gel electrophoresis; and for qac gene carriage by PCR. Characteristics of the nursing homes and their residents were collected from the Medicare Minimum Data Set and Long-Term Care Focus. A total of 829 MRSA isolates were obtained from swabbing 3,806 residents in 26 nursing homes. All isolates had a chlorhexidine MIC of ≤4 μg/ml. Five (0.6%) isolates harbored the qacA and/or qacB gene loci. Mupirocin resistance was identified in 101 (12%) isolates, with 78 (9%) isolates exhibiting high-level mupirocin resistance (HLMR). HLMR rates per facility ranged from 0 to 31%. None of the isolates with HLMR displayed qacA or qacB, while two isolates carried qacA and exhibited low-level mupirocin resistance. Detection of HLMR was associated with having a multidrug-resistant MRSA isolate (odds ratio [OR], 2.69; P = 0.004), a history of MRSA (OR, 2.34; P < 0.001), and dependency in activities of daily living (OR, 1.25; P = 0.004). In some facilities, HLMR was found in nearly one-third of MRSA isolates. These findings may have implications for the increasingly widespread practice of MRSA decolonization using intranasal mupirocin.

Salicylidene acylhydrazides belong to a class of compounds shown to inhibit bacterial type III secretion (T3S) in pathogenic Gram-negative bacteria. This class of compounds also inhibits growth and replication of Chlamydiae, strict intracellular bacteria that possess a T3S system. In this study a library of 58 salicylidene acylhydrazides was screened to identify inhibitors of Chlamydia growth. Compounds inhibiting growth of both Chlamydia trachomatis and Chlamydophila pneumoniae were tested for cell toxicity and seven compounds were selected for preliminary pharmacokinetic analysis in mice using cassette dosing. Two compounds, ME0177 and ME0192, were further investigated by individual pharmacokinetic analysis. Compound ME0177 had a relatively high peak plasma concentration (C(max)) and area under curve and therefore may be considered for systemic treatment of Chlamydia infections. The other compound, ME0192, had poor pharmacokinetic properties but the highest anti-chlamydial activity in vitro and therefore was tested for topical treatment in a mouse vaginal infection model. ME0192 administered vaginally significantly reduced the infectious burden of C. trachomatis and the number of infected mice.

Type III secretion (T3S) is important for the establishment and maintenance of a chlamydial infection. The genes encoding T3S components in Chlamydia are transcribed as separate temporal classes, but the mechanisms that regulate the timing of their expression are not understood. In this study, we demonstrate that promoters for 10 predicted T3S transcriptional units are each transcribed in vitro by the major form of chlamydial RNA polymerase but not by an alternative form of RNA polymerase containing [[sigma].sup.28]. Since changes in DNA supercoiling during chlamydial development have been proposed as a mechanism for temporal gene regulation, we examined the in vitro response of T3S promoters to altered superhelical density. Promoters for three T3S genes that are upregulated at mid times were activated in response to increased DNA supercoiling. In contrast, promoters for three late T3S genes were not sensitive to changes in superhelical density. This differential response to changes in DNA topology is similar to the pattern that has been reported for representative mid and late chlamydial genes that are unrelated to the T3S system. Based on these results, we propose that the temporal expression of T3S genes in Chlamydia is controlled by general mechanisms that regulate [[sigma].sup.66]-dependent gene expression during the developmental cycle. Our results are consistent with a model in which T3S genes that are upregulated in mid cycle are activated together with other mid genes in response to increased DNA supercoiling. doi: 10.1128/JB.00068-10

Chlamydia trachomatis is a major pathogen throughout the world, and preventive measures have focused on the production of a vaccine using the major outer membrane protein (MOMP). Here, in elementary bodies and in preparations of the outer membrane, we identified native trimers of the MOMP. The trimers were stable under reducing conditions, although disulfide bonds appear to be present between the monomers of a trimer and between trimers. Cross-linking of the outer membrane complex demonstrated that the MOMP is most likely not in a close spatial relationship with the 60- and 12-kDa cysteine-rich proteins. Extraction of the MOMP from Chlamydia isolates under nondenaturing conditions yielded the trimeric conformation of this protein as shown by cross-linking and analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with different concentrations of acrylamide. Using circular dichroism spectroscopy, we determined that the trimers were formed mainly of [beta]-pleated sheet structures in detergent micelles. Using a liposomal swelling assay, the MOMP was found to have porin activity, and the size of the pore was estimated to be approximately 2 nm in diameter. The trimers were found to be stable in SDS at temperatures ranging from 4 to 37[degrees]C and over a pH range of 5.0 to 8.0. In addition, the trimers of MOMP were found to be resistant to digestion with trypsin. In conclusion, these results show that the native conformation of the MOMP of C. trachomatis is a trimer with predominantly a [beta]-sheet structure and porin function.

Members of the family Chlamydiaceae possess at least 13 genes, distributed throughout the chromosome, that are homologous with genes of known type III secretion systems (TTS). The aim of this study was to use putative TTS proteins of Chlamydophila pneumoniae, whose equivalents in other bacterial TTS function as chaperones, to identify interactions between chlamydial proteins. Using the BacterioMatch Two-Hybrid Vector system (Stratagene, La Jolla, Calif.), lcrH-2 and sycE, positions 1021 and 0325, respectively, from C. pneumoniae CM-1 were used as 'bait' to identify target genes (positions 0324, 0705, 0708, 0808 to 0810, 1016 to 1020, and 1022) in close proximity on the chromosome. Interaction between the products of the lcrH-2 (1021) and lcrE (copN) (0324) genes was detected and confirmed by pull-down experiments and enzyme immunoassays using recombinant LcrH-2 and LcrE. As further confirmation of this interaction, the homologous genes from Chlamydia trachomatis, serovar E, and Chlamydophila psittaci, Texas turkey, were also cloned in the two-hybrid system to determine if LcrH-2 and LcrE would interact with their orthologs in other species. Consistent with their genetic relatedness, LcrH-2 from C. pneumoniae interacted with LcrE produced from the three species of Chlamydiaceae; LcrH-2 from C. psittaci reacted with LcrE from C. pneumoniae but not from C. trachomatis; and C. trachomatis LcrH-2 did not react with LcrE from the other two species. Deletions from the N and C termini of LcrE from C. pneumoniae identified the 50 C-terminal amino acids as essential for the interaction with LcrH-2. Thus, it appears that in the Chlamydiaceae TTS, LcrH-2 interacts with LcrE, and therefore it may serve as a chaperone for this protein.