A Component of the Xanthomonadaceae Type IV Secretion System Combines a VirB7 Motif with a N0 Domain Found in Outer Membrane Transport Proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 angstrom X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7(XAC2622)) and its interaction with VirB9. NMR solution studies show that residues 27-41 of the disordered flexible N-terminal region of VirB7(XAC2622) interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7(XAC2622) has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7(XAC2622) oligomerizes through interactions involving conserved residues in the N0 domain and residues 42-49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB(7XAC2622) oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.

Surveillance Programme for Healthcare Associated Infections in the State of Sao Paulo, Brazil. Implementation and the first three years` results

Governmental programmes should be developed to collect and analyse data on healthcare associated infections (HAIs). This study describes the healthcare setting and both the implementation and preliminary results of the Programme for Surveillance of Healthcare Associated Infections in the State of Sao Paulo (PSHAISP), Brazil, from 2004 to 2006. Characterisation of the healthcare settings was carried out using a national database. The PSHAISP was implemented using components for acute care hospitals (ACH) or long term care facilities (LTCF). The components for surveillance in ACHs were surgical unit, intensive care unit and high risk nursery. The infections included in the surveillance were surgical site infection in clean surgery, pneumonia, urinary tract infection and device-associated bloodstream infections. Regarding the LTCF component, pneumonia, scabies and gastroenteritis in all inpatients were reported. In the first year of the programme there were 457 participating healthcare settings, representing 51.1% of the hospitals registered in the national database. Data obtained in this study are the initial results and have already been used for education in both surveillance and the prevention of HAI. The results of the PSHAISP show that it is feasible to collect data from a large number of hospitals. This will assist the State of Sao Paulo in assessing the impact of interventions and in resource allocation. (C) 2010 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

Evaluation of biologic occupational risk control practices: Quality indicators development and validation

Background: There is growing demand for the adoption of qualification systems for health care practices. This study is aimed at describing the development and validation of indicators for evaluation of biologic occupational risk control programs. Methods: The study involved 3 stages: (1) setting up a research team, (2) development of indicators, and (3) validation of the indicators by a team of specialists recruited to validate each attribute of the developed indicators. The content validation method was used for the validation, and a psychometric scale was developed for the specialists` assessment. A consensus technique was used, and every attribute that obtained a Content Validity Index of at least 0.75 was approved. Results: Eight indicators were developed for the evaluation of the biologic occupational risk prevention program, with emphasis on accidents caused by sharp instruments and occupational tuberculosis prevention. The indicators included evaluation of the structure, process, and results at the prevention and biologic risk control levels. The majority of indicators achieved a favorable consensus regarding all validated attributes. Conclusion: The developed indicators were considered validated, and the method used for construction and validation proved to be effective.

Care during the continuation phase

As the patient`s treatment progresses, symptoms start to disappear and he or she becomes more familiar with the treatment. The standards in this section focus on the types of elements that need to be considered as the patient progresses from the intensive to the continuation phase of tuberculosis (TB) treatment, leading to less contact with the TB service and a resumption of `normal` activities. Social and psychological as well as physical factors need to be assessed to plan effective care and treatment for the continuation phase. Treatment for TB takes a minimum of 6 months, during which changes to the regimen and personal changes associated with making a recovery can create barriers to continuation of treatment. Lifestyle and other changes that may occur during 6 months of anybody`s life can complicate or be complicated by TB treatment. The patient may move to another location at any point during the course of treatment, in which case it may be necessary to transfer his or her care to another TB management unit. This process needs to be carefully managed to maintain contact with the patient and avoid any break in treatment; this is covered by the third standard in this chapter.

Care during the intensive phase: promotion of adherence

The standards in this chapter focus on maximising the patient`s ability to adhere to the treatment prescribed. Many people are extremely shocked when they are told they have TB, some refuse to accept it and others are relieved to find out what is wrong and that treatment is available. The reaction depends on many factors, including cultural beliefs and values, previous experience and knowledge of the disease. Even though TB is more common among vulnerable groups, it can affect anyone and it is important for patients to be able to discuss their concerns in relation to their own individual context. The cure for TB relies on the patient receiving a full, uninterrupted course of treatment, which can only be achieved if the patient and the health service work together. A system needs to be in place to trace patients who miss their appointments for treatment (late patients). The best success will be achieved through the use of flexible, innovative and individualised approaches. The treatment and care the patient has received will inevitably have an impact on his or her willingness to attend in the future. A well-defined system of late patient tracing is mandatory in all situations. However, when the rates are high (above 10%), any tracing system will be useless without also examining the service as a whole.

Starting treatment: caring for patients and their families

The standards presented in this section focus on providing physical, social and psychological care for the patient at the point he or she is diagnosed with tuberculosis (TB) and starts treatment. Detailed guidance is included with regard to organising directly observed treatment (DOT) safely and acceptably for both the patient and the management unit. The aim is to give the patient the best possible chance of successfully completing treatment according to a regimen recommended by the World Health Organization. If the health service where the patient is diagnosed cannot offer ongoing treatment and care due to a lack of facilities, overcrowding or inaccessibility, the patient needs to be referred to a designated TB management unit (BMU) elsewhere. The patient may also receive treatment from a facility outside a BMU. However care is organised, it is essential for all patients who are diagnosed with TB to be registered at an appropriate BMU so that their progress can be routinely monitored and programme performance can be assessed. To avoid the risk of losing contact with the patient at any stage of their care, good communication is essential between all parties involved, from the patient him/herself to the person supervising their DOT to the BMU.

City tuberculosis control coordinators` perspectives of patient adherence to DOT in Sao Paulo State, Brazil, 2005

SETTING: Thirty-six priority cities in Sao Paulo State, Brazil, with a high incidence of tuberculosis (TB) cases, deaths and treatment default. OBJECTIVE: To identify the perspectives of city TB control coordinators regarding the most important components of adherence strategies adopted by health care teams to ensure patient adherence in 36 priority cities in the State of Sao Paulo, Brazil. DESIGN: Qualitative research with semi-structured interviews conducted with the coordinators of the National TB Control Programme involved in the management of TB treatment services in the public sector. RESULTS: The main issues thought to influence adherence to directly observed treatment (DOT) by coordinators include incentives and benefits delivered to patients, patient-health care worker bonding and comprehensive care, the encouragement given by others to follow treatment (family, neighbours and health professionals), and help provided by health professionals for patients to recover their self-esteem. CONCLUSION: The main aspects mentioned by city TB control coordinators regarding patient adherence to treatment and to DOT in Sao Paulo are improvements in communications, relationships based on trust, a humane approach and including the patients in the decision-making process concerning their health.

Identifying an active case of tuberculosis

The best practice standards set out in chapter 2 of the Best Practice guide focus on the various aspects of identifying an active case of TB and aim to address some of the challenges associated with case detection. The importance of developing a good relationship with the patient from the start, when he or she is often most vulnerable, is emphasised. The first standard focuses on the assessment of someone who might have TB and the second gives detailed guidance about the collection of sputum for diagnosis. The standards are aimed at the health care worker, who assesses the patient when he or she presents at a health care facility and therefore needs to be familiar with the signs, symptoms and risk factors associated with TB. Having suspected TB, the health care worker then needs to ensure that the correct tests are ordered and procedures are followed so that the best quality samples possible are sent to the laboratory and all documentation is filled out clearly and correctly. The successful implementation of these standards can be measured by the accurate and prompt reporting of results, the registration of every case detected and the continued attendance of every patient who needs treatment.

Guidance for the implementation of best practice for the care of patients with tuberculosis

The first two chapters of Best practice for the care of patients with tuberculosis: a guide for low-income countries include an introduction and guidance regarding implementation of best practice. The background to how the guide was developed is significant, as it was developed in collaboration with nurses and other health workers working in the most challenging settings. It therefore provides realistic and practical guidance for best practice where patient loads are large and resources are stretched. Guidance regarding standard setting and clinical audit is an important part of enabling people to recognise the strengths that already exist in their practice and approach those areas that require change in a systematic and practical way. The guide itself consists of a series of standards covering different aspects of patient care, from the moment they seek health care with symptoms to their diagnosis to early stages of treatment, directly observed treatment, the continuation phase and transfer of treatment. There are also standards relating specifically to HIV testing and the care of patients co-infected with tuberculosis and HIV. The standards themselves will appear in full in the subsequent chapters of this series.

HIV testing and care of the patient co-infected with tuberculosis and HIV

Human immunodeficiency virus (HIV) infection poses one of the greatest challenges to tuberculosis (TB) control, with TB killing more people with HIV infection than any other condition. The standards in this chapter cover provider-initiated HIV counselling and testing and the care of HIV-infected patients with TB. All TB patients who have not previously been diagnosed with HIV infection should be encouraged to have an HIV test. Failing to do so is to deny people access to the care and treatment they might need, especially in the context of the wider availability of treatments that prevent infections associated with HIV A clearly defined plan of care for those found to be co-infected with TB and HIV should be in place., with procedures to ensure that the patient has access to this care before offering routine testing for HIV in persons with TB. It is acknowledged that people caring for TB patients should ensure that those who are HIV positive are transferred for the appropriate ongoing care once their TB treatment has been completed. In some cases, referral for specialised HIV-related treatment and care may be necessary during treatment for TB. The aim of these standards is to enable patients to remain as healthy as possible, whatever their HIV status.

Listeria monocytogenes and Salmonella spp. in raw milk produced in Brazil: Occurrence and interference of indigenous microbiota in their isolation and development

This study aimed to verify the occurrence of Listeria monocytogenes and Salmonella spp. in raw milk produced in Brazil. On account of the poor microbiological quality of this product, possible interference from the indigenous microbiota in these pathogens was also evaluated. Two-hundred and ten raw milk samples were collected in four important milk-producing areas in Brazil, tested for L. monocytogenes and Salmonella spp. presence, and for enumeration of indicator microorganisms: mesophilic aerobes, total coliforms and Escherichia coli. The interference of the indigenous microbiota in the isolation procedures was also tested, as well the frequency of naturally occurring raw milk strains with antagonistic activity against both pathogens. The pathogens were not isolated in any raw milk sample, but poor microbiological quality was confirmed by the high levels of indicator microorganisms. When present at high levels, the indigenous microbiota generated an evident interference in the methodologies of L. monocytogenes and Salmonella spp. isolation, mainly when the pathogens appeared at low levels. Three-hundred and sixty raw milk strains were tested for antagonistic activity against both pathogens, and 91 (25.3%) showed inhibitory activity against L. monocytogenes and 33 (9.2%) against Salmonella spp. The majority of the antagonistic strains were identified as Lactic Acid Bacteria species, mainly Lactococcus lactis subsp. lactis and Enterococcus faecium, known by antimicrobial substance production.

Rapid detection of resistance to pyrazinamide in Mycobacterium tuberculosis using the resazurin microtitre assay

Objectives: The resazurin microtitre plate assay (REMA) was evaluated to determine the susceptibility of Mycobacterium tuberculosis to pyrazinamide, and was compared with the broth microdilution method (BMM), the absolute concentration method (ACM) and pyrazinamidase (PZase) determination. Methods: Thirty-four M. tuberculosis clinical isolates (26 susceptible and 8 resistant to pyrazinamide) and reference strains M. tuberculosis H37Rv ATCC 27294 and Mycobacterium bovis AN5 were tested. Results: REMA and BMM showed 100% specificity and sensitivity when compared with ACM; BMM, however, demanded more reading time. The PZase determination assay showed 87.50% and 100% sensitivity and specificity, respectively. Conclusions: All tested methods in this preliminary study showed excellent sensitivity and specificity for the determination of pyrazinamide susceptibility of M. tuberculosis, but REMA was faster, low-cost and easy to perform and interpret. Additional studies evaluating REMA for differentiating pyrazinamide-resistant and-susceptible M. tuberculosis should be conducted on an extended panel of clinical isolates.

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

P>Thirty-five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Parana, Brazil. Twenty-two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff`s method and processed for acid-fast bacilli staining, culture in Stonebrink and Lowenstein-Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR-positive results (54.5%) was similar to that of culture-positive results (51.5%, P > 0.05). The percentage of PCR-positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR-negative were reanalysed using 2.5 mu l DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.

Virulence features of atypical enteropathogenic Escherichia coli identified by the eae(+) EAF-negative stx(-) genetic profile

This study characterized 76 atypical enteropathogenic Escherichia coli (aEPEC) strains, previously classified by the eae(+) EAF-negative stx(-) genotype, isolated from children with diarrhea in Brazil. Presence of bfpA and bfpA/perA was detected in 2 and 6 strains, respectively. The expression of bundle-forming pilus (BFP), however, was observed by immunofluorescence in 1 bfpA and 3 bfpA/perA strains, classifying them as typical EPEC (tEPEC). The remaining 72 aEPEC strains were characterized by serotyping, intimin typing, adherence patterns to HEp-2 cells, capacity to induce actin aggregation (fluorescent actin staining test), and antimicrobial resistance. Our results show that aEPEC comprise a very heterogeneous group that does not present any prevalence or association regarding the studied characteristics. It also suggest that tEPEC and aEPEC must not be classified only by the reactivity with the EAF probe, and that the search of other markers present in pEAF, as well as the BFP expression, must be considered for this matter. (C) 2009 Elsevier Inc. All rights reserved.

Dissemination of bla(IMP-1)-carrying integron In86 among Klebsiella pneumoniae isolates harboring a new trimethoprim resistance gene dfr23

The genetic context of the bla(IMP-1) gene was evaluated in 9 Klebsiella pneumoniae isolates recovered from 2 hospitals in Sao Paulo, Brazil. All isolates harbored a copy of In86 carrying bla(Imp-1), aac(6`)-31, and aadAl. Eight strains from the same hospital also carried another class I integron harboring a new trimethoprim resistance gene (dfr23) that was chromosomally embedded. In86 was likely to be in a 30-kb nontransferable plasmid and was flanked upstream by a sequence identical to one identified in an IMP-1-producing Pseudomonas putida isolate. The bla(IMP-1)-carrying integron In86 was recently reported from nonfermentative bacilli isolated in Sao Paulo. These isolates appear to be the Source of this integron now acquired by K. pneumoniae strains from different hospitals in the same city. Metallo-beta-lactamase production is still rare among Enterobacteriaceae isolates in Brazil, but the acquisition of genetic structures carrying these mobile resistance determinants is worrisome and could lead to an increase in the prevalence of these phenotypes of resistance. (C) 2008 Elsevier Inc. All rights reserved.