Group A Streptococcus (GAS) causes the life-threatening infection in humans known as necrotizing fasciitis (NF). Infected subcutaneous tissues from an NF patient and mice challenged with the same GAS strain possessed high bacterial loads but a striking paucity of infiltrating polymorphonuclear leukocytes (PMNs). Impaired PMN recruitment was attributed to degradation of the chemokine IL-8 by a GAS serine peptidase. Here, we use bioinformatics approach coupled with target mutagenesis to identify this peptidase as ScpC. We show that SilCR pheromone downregulates scpC transcription via the two-component system-SilA/B. In addition, we demonstrate that in vitro, ScpC degrades the CXC chemokines: IL-8 (human), KC, and MIP-2 (both murine). Furthermore, using a murine model of human NF, we demonstrate that ScpC, but not the C5a peptidase ScpA, is an essential virulence factor. An ScpC-deficient mutant is innocuous for untreated mice but lethal for PMN-depleted mice. ScpC degrades KC and MIP-2 locally in the infected skin tissues, inhibiting PMN recruitment. In conclusion, ScpC represents a novel GAS virulence factor functioning to directly inactivate a key element of the host innate immune response.

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SummaryGroup A streptococcus (GAS) causes diseases ranging from benign to severe infections such as necrotizing fasciitis (NF). The reasons for the differences in severity of streptococcal infections are unexplained. We developed the polymorphic-tag-lengthstransposon-mutagenesis (PTTM) method to identify virulence genes in vivo . We applied PTTM on an emm 14 strain isolated from a patient with NF and screened for mutants of decreased virulence, using a mouse model of human soft-tissue infection. A mutant that survived in the skin but was attenuated in its ability to reach the spleen and to cause a lethal infection was identified. The transposon was inserted into a small open reading frame (ORF) in a locus termed sil , s treptococcal i nvasion l ocus. sil contains at least five genes ( sil A-E) and is highly homologous to the quorum-sensing competence regulons of Streptococcus pneumoniae . sil A and sil B encode a putative two-component system whereas sil D and sil E encode two putative ABC transporters. sil C is a small ORF of unknown function preceded by a combox promoter. Insertion and deletion mutants of sil had a diminished lethality in the animal model. Virulence of a deletion mutant of sil C was restored when injected together with the avirulent emm 14-deletion mutant, but not when these mutants were injected into opposite flanks of a mouse. DNA transfer between these mutants occurred in vivo but could not account for the complementation of virulence. DNA exchange between the emm 14-deletion mutant and mutants of sil occurred also in vitro, at a frequency of ~ 10 ----8 for a single antibiotic marker. Whereas sil C and sil D mutants exchanged markers with the emm 14 mutant, sil B mutant did not. Thus, we identified a novel locus, which controls GAS spreading into deeper tissues and could be involved in DNA transfer.

Streptococcus pyogenes (group A streptococcus [GAS]) is a frequent cause of purulent infections in humans. As potentially important aspects of its pathogenicity, GAS was recently shown to aggregate, form intratissue microcolonies, and potentially participate in multispecies biofilms. In this study, we show that GAS in fact forms monospecies biofilms in vitro, and we analyze the basic parameters of S. pyogenes in vitro biofilm formation, using Streptococcus epidermidis as a biofilm-positive control. Of nine clinically important serotype strains, M2, M6, M14, and M18 were found to significantly adhere to coated and uncoated polystyrene surfaces. Fibronectin and collagen types I and IV best supported primary adherence of serotype M2 and M18 strains, respectively, whereas serotype M6 and M14 strains strongly bound to uncoated polystyrene surfaces. Absorption measurements of safranin staining, as well as electron scanning and confocal laser scanning microscopy, documented that primary adherence led to subsequent formation of three-dimensional biofilm structures consisting of up to 46 bacterial layers. Of note, GAS isolates belonging to the same serotype were found to be very heterogeneous in their biofilm-forming behavior. Biofilm formation was equally efficient under static and continuous flow conditions and consisted of the classical three steps, including partial disintegration after long-term incubation. Activity of the SilC signaling peptide as a component of a putative quorum-sensing system was found to influence the biofilm structure and density of serotype M14 and M18 strains. Based on the presented methods and results, standardized analyses of GAS biofilms and their impact on GAS pathogenicity are now feasible.

Mycoplasma pneumoniae infections occur both endemically and epidemically, and macrolide resistance has been spreading for 10 years worldwide. A substantial increased incidence of M. pneumoniae infections has been reported in several countries since 2010. Whether this increased incidence is attributed to different or to the same M. pneumoniae genotype is unknown. We have developed a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) for the molecular typing of M. pneumoniae isolates. In this study, the MLVA typing method was modified and validated to be applicable directly to respiratory tract specimens without culture. This method was applied to 34 M. pneumoniae-positive specimens received at the Bordeaux Hospital, France, between 2007 and 2010 in an endemic setting, and to 63 M. pneumoniae-positive specimens collected during an epidemic surge of M. pneumoniae infections in 2010 in Jerusalem, Israel. The M. pneumoniae endemic spread was shown to be polyclonal in France, with 15 MLVA types identified. Strikingly, the Israeli epidemic surge was also a multi-clonal phenomenon, with 18 circulating MLVA types. The macrolide resistance-associated substitution, A2058G, was found in 22% of the Israeli patients. Macrolide-resistant M. pneumoniae belonged to four MLVA types, the MLVA type Z being the most frequent one. An association between the MLVA type Z and macrolide resistance might exist since macrolide resistance was present or generated during the course of illness in all patients infected with this MLVA type. In conclusion, the discriminatory power of the MLVA showed that the spread of M. pneumoniae strains in France in an endemic setting was polyclonal as well as the surge of M. pneumoniae infections in Israel in 2010.

Macrolide resistance in Mycoplasma pneumoniae is often found in Asia but is rare elsewhere. We report the emergence of macrolide-resistant M. pneumoniae in Israel and the in vivo evolution of such resistance during the treatment of a 6-year-old boy with pneumonia.M ycoplasma pneumoniae is a leading respiratory pathogen in both pediatric (1,2) and adult (1,3) populations. Macrolides are considered the fi rst line of therapy and are almost the only treatment for children. In recent years, alarming rates of M. pneumoniae with macrolide resistance (<90%) have occurred in eastern Asia, including the People's Republic of China, Japan, and Korea (2,4-7). This was initially reported in children; however, a surge of resistance in adults was recently reported (2,4,7). Macrolide-resistant M. pneumoniae has also been suggested to be associated with a longer course of disease (2,4).In the Western Hemisphere, lower rates of macrolide resistance have been reported (<10%), however, several epidemics with notable complications have occurred (8)(9)(10)(11). We report the detection of macrolide resistance in M. pneumoniae in Israel. The StudyA previously healthy 6-year-old boy was hospitalized after 2 weeks with fever up to 40°C. At onset of illness, a diagnosis of pharyngitis was made. Streptococcus pyogenes was isolated from his throat, and amoxicillin was prescribed without any clinical response. Later, a clinical diagnosis of sinusitis was made, and amoxicillin-clavulanate was prescribed. A chest radiograph done at that time reportedly showed no abnormalities. Laboratory investigation before admission showed leukocytosis of 19,600 cells/mm 3 with 2,200 monocytes/mm 3 and 7,600 neutrophils/mm 3 ; L-lactate dehydrogenase (LDH) was 1,854 U/L (reference value up to 600 U/L).Ten days after the beginning of his illness, his fever decreased for 2 days and then reappeared, together with cough, resulting in hospitalization. At admission, pneumonia of the right middle and lower lobe was confi rmed by chest radiograph. Laboratory tests showed leukocytes within normal ranges, erythrocyte sedimentation rate (ESR) 80/h, and C-reactive protein (CRP) 15 mg/L (reference range up to 0.5 mg/L). Treatment with penicillin was started without clinical improvement. Azithromycin (10 mg/kg/d) was added on the third day. After receiving this treatment, his leukocytes increased to 20,000 cells/mm 3 with ESR 97/h and CRP 22.5 mg/L. The β-lactam coverage was switched to cefuroxime and later to ceftriaxone because no response was observed. Chest ultrasound showed a small pleural effusion. Bronchoscopy showed thick mucus secretions; respiratory specimens tested were negative for respiratory syncytial virus, infl uenza viruses A and B, parainfl uenza virus, human metapneumovirus, and adenovirus, as were results of urine tests for Legionella spp. and blood tests for pneumococcal antigen and cryptococcal antigen.Throat swab specimens were collected and DNA extracted by boiling. Samples were positive for M. pneumoniae by real-time PCR based on the detection of ...

A single CRKP clone ST512 has spread efficiently in our region. In this clone, aac(6')-Ib, common in CRKP strains, is carried on a different plasmid from bla(KPC-3).

Mycoplasma pneumoniae is a leading cause of respiratory disease. In the Intensive Care Unit (ICU) setting M. pneumoniae is not considered a common pathogen. In 2010-13 an epidemic of M. pneumoniae-associated infections was reported and we observed an increase of M. pneumoniae patients admitted to ICU. We analysed the cohort of all M. pneumoniae-positive patients' admissions during 2007 to 2012 at the Hadassah-Hebrew University Medical Centre (a 1100-bed tertiary medical centre). Mycoplasma pneumoniae diagnosis was made routinely using PCR on throat swabs and other respiratory samples. Clinical parameters were retrospectively extracted. We identified 416 M. pneumoniae-infected patients; of which 68 (16.3%) were admitted to ICU. Of these, 48% (173/416) were paediatric patients with ICU admission rate of 4.6% (8/173). In the 19- to 65-year age group ICU admission rate rose to 18% (32/171), and to 38.8% (28/72) for patients older than 65 years. The mean APACHE II score on ICU admission was 20, with a median ICU stay of 7 days, and median hospital stay of 11.5 days. Of the ICU-admitted patients, 54.4% (37/68) were mechanically ventilated upon ICU admission. In 38.2% (26/68), additional pathogens were identified mostly later as secondary pathogens. A concomitant cardiac manifestation occurred in up to 36.8% (25/68) of patients. The in-hospital mortality was 29.4% (20/68) and correlated with APACHE II score. Contrary to previous reports, a substantial proportion (16.3%) of our M. pneumoniae-infected patients required ICU admission, especially in the adult population, with significant morbidity and mortality.