Allon E. Moses

A locus of group A streptococcus involved in invasive disease and DNA transfer

Group 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‐lengths‐transposon‐mutagenesis (PTTM) method to identify virulence genes in vivo. We applied PTTM on an emm14 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, streptococcal invasion locus. sil contains at least five genes (silA‐E) and is highly homologous to the quorum‐sensing competence regulons of Streptococcus pneumoniae. silA and silB encode a putative two‐component system whereas silD and silE encode two putative ABC transporters. silC 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 silC was restored when injected together with the avirulent emm14‐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 emm14‐deletion mutant and mutants of sil occurred also in vitro, at a frequency of ∼ 10‐8 for a single antibiotic marker. Whereas silC and silD mutants exchanged markers with the emm14 mutant, silB mutant did not. Thus, we identified a novel locus, which controls GAS spreading into deeper tissues and could be involved in DNA transfer.

Group G Streptococcal Bacteremia in Jerusalem

Recurrent group G Steptococcus bacteremia, associated with lymphatic disorders and possibly emm stG840.0, is described.

Human Mast Cell Activation by Staphylococcus aureus: Interleukin-8 and Tumor Necrosis Factor Alpha Release and the Role of Toll-Like Receptor 2 and CD48 Molecules

ABSTRACT The ability of Staphylococcus aureus to invade and survive within host cells is believed to contribute to its propensity to cause persistent and metastatic infections. In addition, S. aureus infections often are associated with atopic diseases such as dermatitis, rhinitis, and asthma. Mast cells, the key cells of allergic diseases, have a pivotal role in innate immunity and have the capacity of phagocytosis, and they can destroy some pathogenic bacteria. However, little is known about the ability of some other bacteria to survive and overcome mast cell phagocytosis. Therefore, we were interested in evaluating the interplay between mast cells and S. aureus. In this study, we show that human cord blood-derived mast cells (CBMC) can be infected by pathogenic S. aureus. S. aureus displayed a high adherence to mast cells as well as invasive and survival abilities within them. However, when infections were performed in the presence of cytochalasin D or when CBMC were preincubated with anti-Toll-like receptor 2 (TLR2) or anti-CD48 antibodies, the invasiveness and the inflammatory response were abrogated, respectively. Furthermore, we observed an increase of TLR2 and CD48 molecules on CBMC after S. aureus infection. The infection of CBMC with S. aureus also caused the release of tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8). Both live and killed S. aureus organisms were found to trigger TNF-α and IL-8 release by CBMC in a time-dependent manner. Cumulatively, these findings suggest that S. aureus internalizes and survives in mast cells. This may play an important role in infections and in atopic diseases associated with S. aureus.

INVASIVE PNEUMOCOCCAL INFECTIONS : A COMPARISON BETWEEN ADULTS AND CHILDREN

&NA; Abbreviations used in this article: CSF, cerebrospinal fluid; HIV, human immunodeficiency virus; IPI, invasive pneumococcal infections; MIC, minimal inhibitory concentration.

Macrolide Resistance in Mycoplasma pneumoniae, Israel, 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.

The spread of Mycoplasma pneumoniae is polyclonal in both an endemic setting in France and in an epidemic setting in Israel.

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.

Expression of different group A streptococcal M proteins in an isogenic background demonstrates diversity in adherence to and invasion of eukaryotic cells.

The M protein of group A streptococcus (GAS) is considered to be a major virulence factor because it renders GAS resistant to phagocytosis and allows bacterial growth in human blood. There are more than 80 known serotypes of M proteins, and protective opsonic antibodies produced during disease in humans are serotype specific. M proteins also mediate bacterial adherence to epithelial cells of skin and pharynx. GAS strains vary in the genomic organization of the mga regulon, which contains the genes encoding M and M‐like proteins and other virulence factors. This diversity of organization makes it difficult to assess virulence of M proteins of different serotypes, unless they can be expressed in an isogenic background. Here, we express M proteins of different serotypes in the M protein‐ and protein F1‐deficient GAS strain, SAM2, which also lacks M‐like proteins. Genes encoding M proteins of different serotypes (emmXs) have been integrated into the SAM2 chromosome in frame with the emm6.1 promoter and its mga regulon, resulting in similar levels of emmX expression. Although SAM2 exhibits a very low level of adherence to and invasion of HEp‐2 and HaCaT cells, a SAM2‐derived strain expressing M6 protein adheres to and invades both cell types. In contrast, the isogenic strain expressing M18 protein adheres to both cell types, but invades with a very low efficiency. A strain expressing M3 protein adheres to both types of cells, but its invasion of HEp‐2 cells is serum dependent. A GAS strain expressing M6 protein does not compete with the isogenic strain expressing M18 protein for adherence to or invasion of HaCaT cells. We conclude that M proteins of different serotypes recognize different repertoires of receptors on the surfaces of eukaryotic cells.

Characterization of a Mouse-Passaged, Highly Encapsulated Variant of Group A Streptococcus in In Vitro and In Vivo Studies

JRS4(HE), a highly encapsulated, mouse-passaged variant of group A streptococcal strain JRS4, was characterized. The mucoid phenotype of JRS4(HE) was preserved after extensive passage in vitro. The level and size of csrRS transcript in JRS4(HE) was similar to that of JRS4, yet JRS4(HE) expressed high levels of has and sagA and exhibited an increased activity of streptolysin S. These findings indicate that the CsrRS repressor system was inactive in JRS4(HE). JRS4(HE) adhered to HEp-2 cells at the stationary phase but did not internalize these cells. At midlogarithmic phase, JRS4(HE) neither adhered to nor internalized cells, because of an increased amount of hyaluronic acid. Mice injected subcutaneously with JRS4(HE) developed large, deep necrotic lesions. In contrast, mice challenged with JRS4 developed small, superficial lesions. Despite the use of a high inoculum, mice challenged with JRS4(HE) did not develop a lethal bacteremic infection. It is concluded that inactivation of CsrRS in vivo is insufficient to cause a spreading necrotic disease.

Ethical Issues Relating to the Use of Antimicrobial Therapy in Older Adults

This article aims to review the literature relating to the ethics of antibiotic prescription decisions in older adults and to offer some suggestions as to how one might approach these difficult problems. According to many studies, most patients and their family members wish to receive antibiotics even when they are terminally ill or suffering from advanced dementia. Health care professionals are also frequently reluctant to deny the use of antibiotics in such situations. We suggest that the difficult decisions regarding whether one should withhold treatment can be based on consideration of the ethical principles of autonomy, beneficence, nonmaleficence, and justice. From the public health point of view, one should also take into account the need to avoid the emergence of antimicrobial resistance, keeping in mind the balance between the benefit to the specific patient and the cost to future patients. Infectious diseases consultants should actively participate in these ethical dilemmas.

Vancomycin-resistant enterococci in long-term care facilities.

Knowledge of the prevalence rates and associated risk markers of vancomycin-resistant enterococci (VRE) colonization among long-term care facility (LTCF) residents could be used to improve screening policies among newly admitted hospital inpatients. In a cross-sectional survey among 1,215 residents of LTCFs in Jerusalem, the VRE carriage rate was 9.6%. Previous hospitalization and antibiotic treatment were associated with elevated VRE colonization rate. In contrast, moderate and severe levels of dependency and prolonged stay in an LTCF were associated with a decrease in the VRE colonization rate.