Staphylococcus aureus Survives with a Minimal Peptidoglycan Synthesis Machine but Sacrifices Virulence and Antibiotic Resistance

Many important cellular processes are performed by molecular machines, composed of multiple proteins that physically interact to execute biological functions. An example is the bacterial peptidoglycan (PG) synthesis machine, responsible for the synthesis of the main component of the cell wall and the target of many contemporary antibiotics. One approach for the identification of essential components of a cellular machine involves the determination of its minimal protein composition. Staphylococcus aureus is a Gram-positive pathogen, renowned for its resistance to many commonly used antibiotics and prevalence in hospitals. Its genome encodes a low number of proteins with PG synthesis activity (9 proteins), when compared to other model organisms, and is therefore a good model for the study of a minimal PG synthesis machine. We deleted seven of the nine genes encoding PG synthesis enzymes from the S. aureus genome without affecting normal growth or cell morphology, generating a strain capable of PG biosynthesis catalyzed only by two penicillin-binding proteins, PBP1 and the bi-functional PBP2. However, multiple PBPs are important in clinically relevant environments, as bacteria with a minimal PG synthesis machinery became highly susceptible to cell wall-targeting antibiotics, host lytic enzymes and displayed impaired virulence in a Drosophila infection model which is dependent on the presence of specific peptidoglycan receptor proteins, namely PGRP-SA. The fact that S. aureus can grow and divide with only two active PG synthesizing enzymes shows that most of these enzymes are redundant in vitro and identifies the minimal PG synthesis machinery of S. aureus. However a complex molecular machine is important in environments other than in vitro growth as the expendable PG synthesis enzymes play an important role in the pathogenicity and antibiotic resistance of S. aureus

Overcoming barriers in evaluating outbreaks of diarrheal disease in resource poor settings: assessment of recurrent outbreaks in Chobe District, Botswana

BACKGROUND: Diarrheal illness remains a leading cause of global morbidity and mortality, with the majority of deaths occurring in children <5 years of age. Lack of resources often prohibits the evaluation of outbreak characteristics and limits progress in managing this important disease syndrome, particularly in Africa. Relying only on existing medical staff and hospital resources, we assess the use of a questionnaire survey tool to identify baseline outbreak characteristics during recurrent diarrheal outbreaks in Chobe, Botswana. METHODS: Using historical surveillance data (2006–2009), the temporal pattern of recurrent diarrheal outbreaks was evaluated among patients <5 years of age presenting to health facilities in Chobe District. Using a questionnaire survey tool, medical staff from selected health facilities assessed patients (all ages) presenting with diarrheal disease during two diarrheal outbreaks (2011–2012). Cluster analysis and classification and regression trees (CART) were used to evaluate patient attributes by outbreak. RESULTS: We identified a bimodal, annual pattern of acute diarrhea in children <5 years of age across years (Wilcox test, W = 456.5, p = 0.052). Historical outbreak periods appeared to coincide with major hydrological phenomena (rainfall/flood recession). Across health facilities, a significant percent of patients in the prospective study were in the ≥5 age class (44%, n = 515 and 35%, n = 333 in the dry and wet season outbreaks, respectively). Cluster analysis of questionnaire data identified two main branches associated with patient age (<5 and ≥5 years of age). Patients did not cluster by outbreak or village. CART examination identified sex and hospitalization as being most predictive of patients <5 years and household diarrhea in patients ≥5 years. Water shortages and water quality deficiencies were identified in both outbreaks. CONCLUSIONS: Diarrhea is a persistent, seasonally occurring disease in Chobe District, Botswana. Lack of variation in outbreak variables suggests the possibility of environmental drivers influencing outbreak dynamics and the potential importance of human-environmental linkages in this region. Public health strategy should be directed at securing improved water service and correcting water quality deficiencies. Public health education should include increased emphasis on sanitation practices when providing care to household members with diarrhea. While global diarrheal disease surveillance is directed at the under-5 age group, this may not be appropriate in areas of high HIV prevalence such as that found in our study area where a large immune-compromised population may warrant increased surveillance across age groups. The approach used in this study provided the first detailed characterization of diarrheal disease outbreaks in the area, an important starting point for immediate intervention and development of working hypotheses for future disease investigations. While data derived from this approach are necessarily limited, they identify critical information on outbreak characteristics in resource poor settings where data gaps continue and disease incidence is high