Antibiotic tolerance and the alternative lifestyles of Staphylococcus aureus.

Staphylococcus aureus has an incredible ability to survive, either by adapting to environmental conditions or defending against exogenous stress. Although there are certainly important genetic traits, in part this ability is provided by the breadth of modes of growth S. aureus can adopt. It has been proposed that while within their host, S. aureus survives host-generated and therapeutic antimicrobial stress via alternative lifestyles: a persister sub-population, through biofilm growth on host tissue or by growing as small colony variants (SCVs). Key to an understanding of chronic and relapsing S. aureus infections is determining the molecular basis for its switch to these quasi-dormant lifestyles. In a multicellular biofilm, the metabolically quiescent bacterial community additionally produces a highly protective extracellular polymeric substance (EPS). Furthermore, there are bacteria within a biofilm community that have an altered physiology potentially equivalent to persister cells. Recent studies have directly linked the cellular ATP production by persister cells as their key feature and the basis for their tolerance of a range of antibiotics. In clinical settings, SCVs of S. aureus have been observed for many years; when cultured, these cells form non-pigmented colonies and are approximately ten times smaller than their counterparts. Various genotypic factors have been identified in attempts to characterize S. aureus SCVs and different environmental stresses have been implicated as important inducers.

A full genomic characterization of the development of a stable Small Colony Variant cell-type by a clinical Staphylococcus aureus strain.

A key to persistent and recurrent Staphylococcus aureus infections is its ability to adapt to diverse and toxic conditions. This ability includes a switch into a biofilm or to the quasi-dormant Small Colony Variant (SCV). The development and molecular attributes of SCVs have been difficult to study due to their rapid reversion to their parental cell-type. We recently described the unique induction of a matrix-embedded and stable SCV cell-type in a clinical S. aureus strain (WCH-SK2) by growing the cells with limiting conditions for a prolonged timeframe. Here we further study their characteristics. They possessed an increased viability in the presence of antibiotics compared to their non-SCV form. Their stability implied that there had been genetic changes; we therefore determined both the genome sequence of WCH-SK2 and its stable SCV form at a single base resolution, employing Single Molecular Real-Time (SMRT) sequencing that enabled the methylome to also be determined. The genetic features of WCH-SK2 have been identified; the SCCmec type, the pathogenicity and genetic islands and virulence factors. The genetic changes that had occurred in the stable SCV form were identified; most notably being in MgrA, a global regulator, and RsbU, a phosphoserine phosphatase within the regulatory pathway of the sigma factor SigB. There was a shift in the methylomes of the non-SCV and stable SCV forms. We have also shown a similar induction of this cell-type in other S. aureus strains and performed a genetic comparison to these and other S. aureus genomes. We additionally map RNAseq data to the WCH-SK2 genome in a transcriptomic analysis of the parental, SCV and stable SCV cells. The results from this study represent the unique identification of a suite of epigenetic, genetic and transcriptional factors that are implicated in the switch in S. aureus to its persistent SCV form.

The induction of Staphylococcus aureus biofilm formation or Small Colony Variants is a strain-specific response to host-generated chemical stresses.

Staphylococcus aureus is extremely versatile. It has a capacity to persist within its host by switching to the alternative lifestyles of biofilm or Small Colony Variants (SCV). The induction of this switch has been presumed to be in response to stressed conditions, however the environmental basis has not been thoroughly investigated. We assessed the response of numerous strains to chemicals that are present in human host. There were some that induced a biofilm or SCV phenotype and indeed some inducing both lifestyles. This result illustrates the diversity within a population and a strain-specific adaptation to the presence of host-generated stresses.

Prolonged growth of a clinical Staphylococcus aureus strain selects for a stable small-colony-variant cell type.

An undetermined feature of Staphylococcus aureus pathogenesis is its persistence and then relapse of disease. This has been explained by its switch to alternative lifestyles, mainly as biofilm or small-colony variants (SCVs). Studying the native characteristics of SCVs has been problematic due to their reversion to the parental lifestyle. We have observed that for a number of S. aureus strains as they switch to an SCV lifestyle, there is the formation of an extracellular matrix. We focused our analysis on one strain, WCH-SK2. For bacterial survival in the host, the combination of low nutrients and the prolonged time frame forms a stress that selects for a specific cell type from the population. In this context, we used steady-state growth conditions with low nutrients and a controlled low growth rate for a prolonged time and with methylglyoxal. These conditions induced S. aureus WCH-SK2 into a stable SCV cell type; the cells did not revert after subculturing. Analysis revealed these cells possessed a metabolic and surface profile that was different from those of previously described SCVs or biofilm cells. The extracellular matrix was protein and extracellular DNA but not polysaccharide. The SCV cells induced expression of certain surface proteins (such as Ebh) and synthesis of lantibiotics while downregulating factors that stimulate the immune response (leucocidin, capsule, and carotenoid). Our data reveal cell heterogeneity within an S. aureus population and under conditions that resemble long-term survival in the host have identified a previously unnoticed S. aureus cell type with a distinctive metabolic and molecular profile.

Doppler assessment of the uterine circulation and the clinical behaviour of gestational trophoblastic tumours requiring chemotherapy.

The haemodynamics of the uterine arteries and myometrium were assessed using Doppler ultrasound in forty consecutive patients requiring treatment for invasive mole and choriocarcinoma. The investigations were performed prior to the commencement of chemotherapy and the subjects followed prospectively. The Doppler waveforms from the uterine arteries were analysed using the pulsatility index. It was found that patients with a pulsatility index of 1.1 or less were significantly more likely to develop drug resistance than those with a higher value (P < 0.04). There was no significant association between the pulsatility index and metastatic disease or uterine bleeding. Five out of eight patients who developed drug resistance could have avoided initial inadequate treatment if the Doppler findings were included in the scoring system for selecting chemotherapy for these tumours. It can be concluded that assessment of the uterine arteries using the pulsatility index prior to the treatment of patients with invasive mole and choriocarcinoma is of help in predicting those who will develop drug resistance.

Ultrasonic morphology of the uterus and ovaries after treatment of invasive mole and gestational choriocarcinoma.

Ultrasound imaging of the uterus and ovaries was performed on 41 patients after completion of apparently successful cytotoxic chemotherapy for invasive mole and choriocarcinoma. Uterine volume was calculated and the echopattern of the uterus and ovaries assessed. Forty-nine per cent of subjects had an abnormal uterine appearance and 20% had ovarian theca-lutein cysts. Two patients relapsed on follow-up but no predictive ultrasonic features could be established on uterine or ovarian morphology after chemotherapy.

Preliminary Doppler studies on the uterine artery and myometrium in trophoblastic tumours requiring chemotherapy.

The uterine circulation in 38 patients with invasive mole or choriocarcinoma were investigated using Doppler ultrasound. Twenty-six non-pregnant and 23 pregnant volunteers were assessed in a similar fashion to act as control groups. The pulsatility indices were significantly lower in the patients with trophoblastic disease when compared with the non-pregnant and pregnant groups (Student's t-test; P less than 0.001 and P less than 0.02 respectively). The uterine circulation in persistent trophoblastic diseases shows a characteristic Doppler profile suggesting the presence of large low resistance blood vessels. This may be useful in the initial assessment of these tumours before cytotoxic chemotherapy.

Doppler time velocity waveform studies of the uterine artery and uterus.

This study describes a reliable and reproducible method of obtaining Doppler frequency shift waveforms from the uterine artery based on observations in 26 non-pregnant women. The waveforms were detected easily but direct display of the vessel was inconsistent. The values of the pulsatility index calculated from the waveforms had a mean of 3.25 (SD 0.83). The standard error of the mean was 0.16 and based on this, the 95% reference range was 1.21 to 5.29. The stage of the menstrual cycle did not affect the pulsatility index nor was there any significant difference between subjects who were nulliparous and those who had had previous pregnancies. These results show that Doppler ultrasound can be applied to the non-pregnant uterus and the data presented may be used as a baseline for the study of uterine pathology.

Symptom duration and pathologic staging of colorectal cancer.

A large proportion of patients with colorectal cancer continue to present with relatively advanced and therefore incurable tumours. While delay in diagnosis is often felt to have been a contributing factor, the validity of this has seldom been questioned. Two hundred and eleven consecutive large bowel cancer patients have been prospectively studied with respect to their duration of symptoms and pathologic stage: 7.1% presented with Dukes A tumours and a mean symptom duration of 11.2 months; 38.9% with Dukes B tumours and a mean symptom duration of 4.9 months; 22.3% with Dukes C1 tumours and a mean symptom duration of 5.3 months; 3.8% with Dukes C2 tumours and a mean symptom duration of 3.9 months and 26% with stage D disease and a mean symptom duration of 3.8 months. There was no tendency for a longer symptomatic period in patients with more advanced disease. Indeed the Dukes A patients had a significantly longer duration of symptoms than those with all other stages of disease. It is argued that diagnosis at a more favourable pathologic stage would only be possible by presymptomatic tumour detection.