Assessment of VITEK® MS IVD database V3.0 for identification of Nocardia spp. using two culture media and comparing direct smear and protein extraction procedures.

We assessed the performance of the VITEK® MS IVD V3.0 matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI-ToF MS) V3.0 database for the identification of Nocardia spp. as compared with targeted DNA sequencing. A collection of 222 DNA sequence-defined Nocardia spp. strains encompassing 18 different species present or not in the database was tested. Bromocresol purple agar (BCP) and Columbia agar +5% sheep's blood (COS) culture media were used together with two different preparation steps: direct smear and a "3 attempts" procedure that covered (1) spotting of an extract, (2) new spotting of the same extract, and (3) spotting of a new extract. The direct smear protocol yielded low correct identification rates (≤ 15% for both media) whereas protein extraction yielded correct identification results (> 67% regardless of the media used.). The use of 2 additional attempts using repeat or new extracts increased correct identification rates to 87% and 91% for BCP and COS, respectively. When using the 3 attempts procedure, the best identification results, independent of media types, were obtained for N. farcinica and N. cyriacigeorgica (100%). Identification attempts 2 and 3 allowed to increase the number of correct identifications (BCP, +20%; COS, +13%). The enhancement in performance during attempts 2 and 3 was remarkable for N. abscessus (81% for both media) and low prevalence species (BCP, 70%; COS, 85%). Up to 3.4% and 2.4% of the strains belonging to species present in the database were misidentified with BCP and COS media, respectively. In 1.9% of the cases for BCP and 1.4% for COS, these misidentifications concerned a species belonging to the same phylogenetic complex. Concerning strains that are not claimed in the V3.0 database, N. puris and N. goodfellowi generated "No identification" results and 100% of the strains belonging to N. arthritidis, N.cerradoensis, and N. altamirensis yielded a misidentification within the same phylogenetic complex. Vitek® MS IVD V3.0 is an accurate and useful tool for identification of Nocardia spp.

Does bacteriology laboratory automation reduce time to results and increase quality management?

Due to reductions in financial and human resources, many microbiological laboratories have merged to build very large clinical microbiology laboratories, which allow the use of fully automated laboratory instruments. For clinical chemistry and haematology, automation has reduced the time to results and improved the management of laboratory quality. The aim of this review was to examine whether fully automated laboratory instruments for microbiology can reduce time to results and impact quality management. This study focused on solutions that are currently available, including the BD Kiestra™ Work Cell Automation and Total Lab Automation and the Copan WASPLab(®).

α-Defensins partially protect human neutrophils against Panton-Valentine leukocidin produced by Staphylococcus aureus.

UNLABELLED: α-Defensins produced by neutrophils are important effector molecules of the innate immune system. In addition to their microbicidal effects, α-defensins have the ability to neutralize bacterial toxins. Panton-Valentine leukocidin (PVL) is the hallmark of community-acquired methicillin-resistant Staphylococcus aureus. Staphylococcus aureus that produce PVL are responsible for severe diseases, including necrotizing pneumonia. Polymorphonuclear neutrophils (PMNs) are the target cells of PVL action. The goal of this study was to elucidate the effect of a group of α-defensins known as the human neutrophil peptides (HNPs) on the interactions between LukS-PV and LukF-PV, which compose PVL, and human PMNs. We observed that HNPs bound to both subunits of PVL and significantly decreased PVL pore formation in PMNs, with a maximum inhibition of 27%. When various HNP molecules were tested individually under the same conditions, we observed that HNP3, but not HNP1 or 2, decreased pore formation. Similarly, HNP3 significantly decreased PVL-induced PMN lysis, with a maximum inhibition of 31%. Interestingly, HNPs did not affect LukS-PV LukF-PV oligomerization, LukS-PV LukF-PV binding to PMNs or calcium influx induced by PVL in PMNs. Our results suggest that HNP3 partially protects neutrophils against PVL by interfering with the conformational changes of PVL required to form a functional pore. SIGNIFICANCE AND IMPACT OF THE STUDY: Panton-Valentine leukocidin (PVL) is a pore-forming toxin produced by Staphylococcus aureus, responsible for neutrophil damage and key player of severe staphylococcal diseases. Antimicrobial peptides produced by neutrophils (HNP1-3) neutralize several other bacterial cytotoxins. We examined the impact of human neutrophil peptides (HNPs) on PVL cytotoxicity against human neutrophils and we found that HNPs bind to both LukS and LukF components of PVL, thereby inhibiting pore formation and neutrophil lysis. Our results suggest that HNP3 may impair PVL conformational changes required to form a functional pore and provide insight into the pathogenesis of PVL-related staphylococcal infection, with potential impact on the disease outcome.

Impacts of enterotoxin gene cluster-encoded superantigens on local and systemic experimental Staphylococcus aureus infections.

Staphylococcus aureus is both a component of the normal skin flora and an important pathogen. It expresses a range of recognized and putative virulence factors, such as enterotoxins with superantigenic properties. Several superantigen genes, i.e., seg, sei, selm, seln, and selo, are encoded by the enterotoxin gene cluster (egc), which is found in the majority of S. aureus isolates. Carriage of egc is associated with fitness of S. aureus in the gut microbiota, but it is not known if it contributes to pathogenicity. We constructed egc+ (functional for the seg, selm, and selo genes) and isogenic egc- S. aureus mutants, and investigated their virulence profiles in murine infection models. No effect of egc was seen in a local skin and soft tissue infection model, but in an invasive infection model, increased weight loss was observed after infection with the egc+ as compared to the egc- mutant. Mortality and arthritis were not affected by egc status. Our data suggest that egc has limited effects on the virulence of S. aureus. It may primarily function as a colonization factor increasing commensal fitness, although it might have some aggravating effects on the infection when the bacteria reach the blood.

Renal haemodynamic response to amino acids infusion in an experimental porcine model of septic shock.

BACKGROUND: Acute kidney injury (AKI) is common in sepsis. Treatments allowing maintenance of renal blood flow (RBF) could help to prevent AKI associated with renal hypoperfusion. Amino acids (AA) have been associated with an increase of RBF and glomerular filtration rate (GFR) in several species. The aim of this study was to evaluate the effects of an AA infusion on RBF and GFR in a porcine model of septic shock. METHODS: A total of 17 piglets were randomly assigned into three groups: Sham (Sham, n = 5), sepsis without AA (S-NAA, n = 6), sepsis treated with AA (S-AA, n = 6). Piglets preparation included the placement of ultrasonic transit time flow probes around left renal artery for continuous RBF measurement; ureteral catheters for GFR and urine output evaluation; pulmonary artery catheter for cardiac output (CO) and pulmonary arterial pressure measurements. Mean arterial pressure (MAP) and renal vascular resistance (RVR) were also determined. Septic shock was induced with a live Pseudomonas aeruginosa infusion. Crystalloids, colloids and epinephrine infusion were used to maintain and restore MAP > 60 mmHg and CO > 80% from baseline. RESULTS: Renal haemodynamic did not change significantly in the Sham group, whereas RBF increased slightly in the S-NAA group. Conversely, a significant increase in RVR and a decrease in RBF and GFR were observed in the S-AA group. AA infusion was associated with a higher requirement of epinephrine [340.0 (141.2; 542.5) mg vs. 32.5 (3.8; 65.0) mg in the S-NAA group P = 0.044]. CONCLUSION: An infusion of amino acids impaired renal haemodynamics in this experimental model of septic shock.

Staphylococcal entertotoxins of the enterotoxin gene cluster (egcSEs) induce nitrous oxide- and cytokine dependent tumor cell apoptosis in a broad panel of human tumor cells.

The egcSEs comprise five genetically linked staphylococcal enterotoxins, SEG, SEI, SElM, SElN, and SElO and two pseudotoxins which constitute an operon present in up to 80% of Staphylococcus aureus isolates. A preparation containing these proteins was recently used to treat advanced lung cancer with pleural effusion. We investigated the hypothesis that egcSEs induce nitrous oxide (NO) and associated cytokine production and that these agents may be involved in tumoricidal effects against a broad panel of clinically relevant human tumor cells. Preliminary studies showed that egcSEs and SEA activated T cells (range: 11-25%) in a concentration dependent manner. Peripheral blood mononuclear cells (PBMCs) stimulated with equimolar quantities of egcSEs expressed NO synthase and generated robust levels of nitrite (range: 200-250 µM), a breakdown product of NO; this reaction was inhibited by NG-monomethyl-L-arginine (L-NMMA) (0.3 mM), an NO synthase antagonist. Cell free supernatants (CSFs) of all egcSE-stimulated PBMCs were also equally effective in inducing concentration dependent tumor cell apoptosis in a broad panel of human tumor cells. The latter effect was due in part to the generation of NO and TNF-α since it was significantly abolished by L-NMMA, anti-TNF-α antibodies, respectively, and a combination thereof. A hierarchy of tumor cell sensitivity to these CFSs was as follows: lung carcinoma > osteogenic sarcoma > melanoma > breast carcinoma >neuroblastoma. Notably, SEG induced robust activation of NO/TNFα-dependent tumor cell apoptosis comparable to the other egcSEs and SEA despite TNF-α and IFN-γ levels that were 2 and 8 fold lower, respectively, than the other egcSEs and SEA. Thus, egcSEs produced by S. aureus induce NO synthase and the increased NO formation together with TNF-α appear to contribute to egcSE-mediated apoptosis against a broad panel of human tumor cells.

Epidemiological data of staphylococcal scalded skin syndrome in France from 1997 to 2007 and microbiological characteristics of Staphylococcus aureus associated strains.

Epidemiological data on staphylococcal scalded skin syndromes (SSSS), including bullous impetigo (BI) and generalized exfoliative syndrome (GES), are scarce. To better characterize SSSS and associated Staphylococcus aureus strains, we conducted a retrospective study of 349 cases collected in France between 1997 and 2007 by the National Reference Centre of Staphylococci. Our results showed a stationary evolution of SSSS cases, with a heterogeneous distribution of cases in France. Although notification was not exhaustive, we estimated an incidence of 0.56 cases/year/million inhabitants, in accordance with previous studies conducted in France and Europe, with a median age of 2 years old and sex ratios of 1. A seasonal effect was observed, with a higher GES/BI ratio in autumn compared with other seasons, which could be explained by the impact of viral co-infection. Genetic analysis of S. aureus strains showed that accessory gene regulator (agr) 4, exfoliative toxin A (eta) and B (etb) genes, staphylococcal and enterotoxin-like O (selo) gene and agr4 etb selo profiles were predominantly associated with GES, whereas agr2 eta and agr4 eta selo were more frequently observed with BI. Only one methicillin-resistant strain was found. Protein A (spa) typing identified two main genotypes: spa clonal complex (CC) 159/sequence-type (ST) 121 (75%) and spaCC346/ST15 (18%). spaCC159 was mainly associated with agr4 eta etb selo, agr4 eta selo and agr4 etb selo, and spaCC346 was mainly associated with agr2 eta, suggesting that French SSSS cases are caused by these two main lineages. However, in a multivariate analysis, only etb was independently associated with GES.

Pragmatic management of Panton-Valentine leukocidin-associated staphylococcal diseases.

Panton-Valentine leukocidin (PVL)-producing Staphylococcus aureus is associated with a broad spectrum of diseases, ranging from common uncomplicated soft tissue infections to severe diseases such as complicated soft tissue infections, extensive bone and joint infections, and necrotising pneumonia. Specialised management of infection based on the presence of PVL may not be required for mild infections, whereas it could be lifesaving in other settings. Moreover, most severe PVL diseases are recently identified entities and a 'gold standard' treatment from comparatives studies of different therapeutic options is lacking. Thus, recommendations are based on expert opinions, which are elaborated based on theory, in vitro data and analogies with other toxin-mediated diseases. In this review, we consider the potential need for specialised PVL-based management and, if required, which tools should be used to achieve optimal management.

Species identification of staphylococci by amplification and sequencing of the tuf gene compared to the gap gene and by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Staphylococcal species, notably, coagulase-negative staphylococci (CoNS), are frequently misidentified using phenotypic methods. The partial nucleotide sequences of the tuf and gap genes were determined in 47 reference strains to assess their suitability, practicability, and discriminatory power as target molecules for staphylococcal identification. The partial tuf gene sequence was selected and further assessed with a collection of 186 strains, including 35 species and subspecies. Then, to evaluate the efficacy of this genotyping method versus the technology of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), the 186 strains were identified using MALDI-TOF-MS (Axima® Shimadzu) coupled to the SARAMIS® database (AnagnosTec). The French National Reference Center for Staphylococci identification method was used as a reference. One hundred and eighty-four strains (98.9%) were correctly identified by tuf gene sequencing. Only one strain was misidentified and one was unidentified. MALDI-TOF-MS identified correctly 138 isolates (74.2%). Four strains were misidentified, 39 were unidentified, five were identified at the group (hominis/warneri) level, and one strain was identified at the genus level. These results confirm the value of MALDI-TOF-MS identification for common species in clinical laboratory practice and the value of the partial tuf gene sequence for the identification of all staphylococcal species as required in a reference laboratory.

High prevalence of methicillin-resistant Staphylococcus aureus clone ST80-IV in hospital and community settings in Algiers.

USA300 is an epidemic community-acquired methicillin-resistant Staphylococcus aureus (C-MRSA) clone in the USA, whereas the European C-MRSA clone ST80-IV has mainly a sporadic diffusion in Europe. The prevalence of European clone ST80-IV in Algeria is poorly documented. We prospectively studied S. aureus infections at Mustapha Bacha hospital in Algiers over a 20-month period. S. aureus nasal colonization was studied during a further 6-month period. The European clone ST80-IV was responsible for more than one-third of both community infections (35.7%) and hospital infections (35.8%). Panton-Valentine leukocidin (PVL)-positive MRSA isolated from hospital inpatients were resistant to multiple antibiotics, including fluoroquinolones in 44.9% of cases. The PVL-positive MRSA nasal carriage rate was high among patients and staff in the dermatology unit (8.7% and 18.5%, respectively), but low (2.7%) among patients attending the outpatient clinic. The European PVL-positive C-MRSA clone ST80-IV is widespread in the Algiers hospital and community settings.

[High prevalence of community- and hospital-acquired infections of methicillin-resistant Staphylococcus aureus containing Panton-Valentine leukocidin gene in Algiers]. / Forte prévalence des infections communautaires et nosocomiales à Staphylococus aureus résistant à la méticilline et portant le gène de la leucocidine de Panton-Valentine dans l'Algérois.

OBJECTIVES: To determine the prevalence of community acquired and hospital methicillin-resistant Staphylococcus aureus (S. aureus) infections and the Panton-Valentine leukocidin. PATIENTS AND METHODS: Seven hundred S. aureus strains were collected during 21 months period in Mustapha Bacha hospital. Bacterial identification was based on standard methods and susceptibilities were tested by disk diffusion method. Molecular study (toxins, mecA gene and agr alleles) were determined for 221 S. aureus isolates by multiplex PCR. RESULTS: The global MRSA prevalence was 42 %, 35 % in the community and 49 % in hospital setting. The frequency of strains containing PVL genes (PVL+) was 36 %, their molecular profile was: agr3, mecA+, etd, edin, which correspond to the C-MRSA major ST80 clone in Europe and the Maghreb. The H-MRSA-PVL+ were multidrug resistant. Among the MSSA, 13 strains contained the tst gene and five contained the exfoliatine genes ETA and ETB. CONCLUSION: Our results show a high rate of MRSA-PVL+ in the community and the hospital setting. The H-MRSA-PVL+ were multidrug resistant complicating their antibiotic treatment options.

Serum antibodies against Panton-Valentine leukocidin in a normal population and during Staphylococcus aureus infection.

To determine whether Staphylococcus aureus Panton-Valentine leukocidin (PVL) is expressed during human infection, anti-PVL antibody titres were compared in patients with PVL-positive and PVL-negative staphylococcal infections, and in patients with no evidence of S. aureus infection. Patients with PVL-positive strains had higher levels of anti-PVL antibodies than individuals of both control groups. The median anti-PVL titre increased 8.6-fold during the course of PVL-positive infection and 1.4-fold during PVL-negative infection. These results indicate that only PVL-positive S. aureus strains elicit significant anti-PVL antibody production in humans, and demonstrate the production of PVL during PVL-positive S. aureus infection. The protective role of this immune response remains to be established.

In vivo and in vitro detection of a superantigenic toxin Vbeta signature in two forms of streptococcal toxic shock syndrome.

The aim of this study was to examine the production of superantigenic toxins in vivo and in vitro in two patients with streptococcal toxic shock syndrome (TSS). In the first patient, a woman with puerperal fever and Streptococcus pyogenes peritonitis, flow cytometry of blood cells and in vitro studies of the isolate showed massive expansion of Vbeta 2-positive T cells corresponding to SpeC production. In the second case, involving a patient with streptococcal TSS and purpura fulminans following non-steroidal anti-inflammatory drug (NSAID) therapy, no Vbeta expansion of T cells was observed in vivo, but the SpeC Vbeta signature was also detected in vitro. In this latter patient, NSAID administration and/or severe disseminated infection might partly explain the absence of Vbeta T cell expansion in vivo. Combined in vivo and in vitro detection of a superantigenic toxin Vbeta signature may be useful to determine which superantigenic toxin is involved in individual cases of streptococcal TSS.

Outbreak of staphylococcal bullous impetigo in a maternity ward linked to an asymptomatic healthcare worker.

An outbreak of staphylococcal bullous impetigo occurred over a period of five months in a maternity ward involving seven infected and two colonised neonates. The skin lesions were due to epidermolytic toxin A-producing Staphylococcus aureus. Infection control measures were implemented and a retrospective case-control study performed. Contact with an auxiliary nurse was the only risk factor for cases of bullous impetigo (P<0.01). The nurse cared for all seven cases and was an asymptomatic nasal carrier of the epidemic strain. Repeated courses of decontamination treatment failed to eradicate carriage. Nine months after the last case, another neonate developed a more severe form of bullous impetigo and the auxiliary nurse was reassigned to an adult ward.