Whole-genome sequencing in the investigation of recurrent invasive group A streptococcus outbreaks in a maternity unit.

BACKGROUND: The clinical manifestations of group A streptococcus (GAS) (Streptococcus pyogenes) are diverse, ranging from asymptomatic colonization to devastating invasive disease. Maternity-related clusters of invasive GAS (iGAS) infection are complex to investigate and control, especially if recurrent. AIM: To investigate three episodes of emm 75 GAS/iGAS infection in maternity patients at one hospital site over a four-year period (two with monophyletic ancestry). METHODS: The episodes are described, together with whole-genome sequence (WGS) isolate analyses. Single nucleotide polymorphism differences were compared with contemporaneous emm 75 genomes. FINDINGS: Over the four-year study period, seven mothers had emm 75 GAS/iGAS and one mother had emm 3 iGAS (in year 4) (subsequently discounted as linked). Three (clinical/screening samples) of the seven babies of emm-75-positive mothers and three screened healthcare workers were positive for emm 75 GAS. WGS similarity suggested a shared ancestral lineage and a common source transmission, but directionality of transmission cannot be inferred. However, the findings indicate that persistence of a particular clone in a given setting may be long term. CONCLUSIONS: Occupational health procedures were enhanced, staff were screened, and antibiotic therapy was provided to GAS-positive staff and patients. The definitive source of infection could not be identified, although staff-patient transmission was the most likely route. The pattern of clonal GAS transmission over the four-year study period suggests that long-term persistence of GAS may have occurred.

Random meticillin-resistant Staphylococcus aureus carrier surveillance at a district hospital and the impact of interventions to reduce endemic carriage.

Colonisation with meticillin-resistant Staphylococcus aureus (MRSA) has previously been described as a risk factor for subsequent infection. MRSA colonisation reached endemic proportions in most healthcare institutions in the UK during the 1990s. Bacteraemia due to MRSA is associated with increased mortality and morbidity compared with meticillin-susceptible S. aureus and national targets have been set for reduction. We present our findings of regular random colonisation surveillance and systematic decolonisation of MRSA carriers over a five-year period with the aim of reducing the pool of carriers and number of MRSA bacteraemia cases. Interventions to reduce the rate of colonisation included assurance of decolonisation and follow up, targeting wards with the highest carriage rates using enhanced screening and education, and screening all admissions aged >65 years. There was a statistically significant reduction in the proportion of patients colonised from 14.6% to 7.0% (P<0.001) and the total number of bacteraemia cases from 42 to 22 (P=0.012) in the initial 24 months of surveillance compared to the most recent 24 months. Regular surveillance of MRSA carriage is useful for monitoring the effects of control measures on MRSA carriage among inpatients. Interventions to reduce carriage are able to reduce the pool of MRSA carriers, thereby reducing cases of bacteraemia.

Effect of ethanesulfonic acid buffers and pH on the accumulation of a nervous system-specific protein (S-100) and a glial-enriched enzyme in a clonal line of rat astrocytes (C6).

Stationary phase cultures of a clonal line of rat astrocytes (C6) were maintained at pH values ranging from 6.0 to 8.4 using media buffered with various combinations of organic buffers or graded concentrations of bicarbonate ion at a constant CO2 tension. The accumulation of a soluble acidic protein unique to the nervous system (S-100) in media buffered with organic buffers was optimal in the pH range 6.4 to 6.8, significantly more acid than that optimal for cell growth (pH 7.0 to 7.8). Cells maintained in CO2-bicarbonate-buffered media exhibited a higher and less marked pH optimum for S-100 protein accumulation and a lower efficiency of accumulation of the protein. These data suggest that the organic buffer ions themselves, apart from their function as buffers, are influencing the accumulation of S-100. The specific activity (assayed at the enzymatic pH optimum) of a membrane-bound enzyme enriched in glial cells and myelin, 2',3'-cyclic nucleotide 3'-phosphohydrolase, was markedly pH-dependent. The optimal pH range was 6.4 to 6.7 in organic buffer controlled media. In CO2-bicarbonate controlled media the optimal pH range was only slightly higher (pH 6.6 to 7.0), but the specific activities were reduced relative to organic buffer-grown cells. The structural relationship of some of the aminoethanesulfonic acid buffers used in these experiments to certain compounds of neurochemical interest (such as taurine and alpha-flupenthixol) is noted.

Some paradoxical effects of inhibitors of protein synthesis on protein turnover in cultured human cells.

Low concentrations of cycloheximide, sufficient to block net protein synthesis in growing normal and cancer cells, had no effect on protein turnover, i.e. either the incorporation of labeled amino acids from media lacking other amino acids essential for growth, or the loss to the medium of amino acids from prelabeled cells. At the concentrations that blocked growth, the rate of amino acid incorporation from complete medium was reduced to the "turnover level" i.e. the rate of incorporation seen in amino acid-deficient media. Protein turnover was inhibited only at higher concentrations of the inhibitor. Qualitatively similar results have been obtained with puromycin, anisomycin, emetin and tylocerebrine.

Effect of environmental pH on rescue of Simian virus 40.

Rescue of SV40 virus after Sendai virus-mediated fusion of transformed mouse or hamster cell lines with permissive monkey cells was strikingly dependent on pH in the range 6.4-8.8, with a maximum at pH 8.4. The titer of virus recovered at pH 8.4 was 2 logs higher than that at pH 7.6, and 4 logs higher than that at pH 6.4. The pH-sensitive step was neither the number of heterokaryocytes formed, which was essentially the same at pH 7.6 and 8.4, nor the degree of SV40 replication in the monkey cells, which was also unaffected by pH variation in the range 7.2-8.4.

Effect of environmental pH on the efficiency of cellular hybridization.

The hybridization of a human and mouse cell was strikingly pH-dependent, with a well defined optimum at (about) pH 7.6-8.0. The yield of hybrid cell colonies (1 per 500-2000 heterokaryocytes) was several hundred times greater than that obtained at pH 6.8-7.2. Although there was a significant effect on the efficiency of cell fusion, the critical time for the pH effect was in the first 4-8 days after fusion, presumably while viable hybrids were being formed from the multinucleated heterokaryocytes.

Buffer combinations for mammalian cell culture.

The growth and metabolism of cultured mammalian cells are markedly affected by the pH variation in ordinary hicarbonate-buffered media (pH 8.0 to 6.9). Those pH swings can be reduced and the pH of the culture can be stabilized as desired in the range pH 6.4 to 8.3 by appropriate combinations of two or three organic buffers, each at 10 to 15 millimolar, in conjunction with phosphate and bicarbonate. The initial alkalinization in sparse cultures is then minimized, and the metabolic acidificatiotn in 24 hours is usually less than 0.4 pH unit except in heavy cultutres.

pH as a determinant of cellular growth and contact inhibition.

1) Both the growth rate and the maximum population density of several normal, virus-transformed, and cancer cells were markedly pH-dependent; the optimum varied from pH 6.9 to 7.8. At the optimum pH, some diploid human cells attained population densities comparable to those of cancer or virus-transformed cells. Contact inhibition of growth is facilitated by repeated fluctuations of pH in nonphysiological ranges, and may not be an intrinsic and necessary attribute of diploid cells in culture. 2) At pH 8.3, at which there was little or no cellular multiplication, the protein content per cell increased 2- to 5-fold over a period of 10-16 days, and was slowly reversed to normal concentrations on restoration of pH to the optimal range. 3) Uridine uptake by contact-inhibited human cell cultures was stimulated by refeeding with salt solution, and to the same extent as by complete (serum-supplemented) growth medium; that immediate increase did not involve the reinitiation of cellular growth and multiplication. Contact inhibition was, however, reversed in 2-4 days by an appropriate increase in the serum concentration of the medium.

Morphologic changes accompanying senescence of cultured human diploid cells.

The lysosomes of serially propagated human fibroblasts gradually transform to residual bodies which increase in number and size, and show progressive degenerative changes. There is an accompanying, and less regular, decrease in the number of cytoplasmic polyribosomes and an increased number of glycogen particles. The onset of these morphologic alterations occurs shortly after culture initiation and precedes any marked decrease in the rate of cellular growth; however, in their extreme form these changes may be related to the ultimate cessation of cellular multiplication ("senescence"). The lysosomal changes were seen only in those cell strains which eventually showed senescence, and were absent or minimal either in cell lines which can be propagated indefinitely ("spontaneous" and viral transformants, cancer cells), or in skin sections from aging subjects.

Growth characteristics of virus-transformed cells. Maximum population density, inhibition by normal cells, serum requirement, growth in soft agar, and xenogeneic transplantability.

Virus transformants (like cancer cells, cells transformed by X-ray or carcinogens, or those which have transformed spontaneously) exhibit a number of phenotypic changes which are usually associated, and which may be lost concurrently. That association is, however, not invariable. More particularly, the altered characteristics here studied (escape from contact inhibition of growth and susceptibility to inhibition by other cells, decreased serum requirement, and ability to grow in soft agar) do not, in and of themselves, endow the cell with the capacity to produce a tumor, at least as judged by the methods of assay here used. Although the question as to whether the tumorigenicity of virus transformants is causally linked to any of these associated changes cannot be answered definitively, the evidence suggests a close linkage, rather than identity, between the determinants of oncogenicity and the other properties here studied.

Evidence for intergenic complementation in hybrid cells derived from two human diploid strains each carrying an X-linked mutation.

Two male diploid fibroblast strains, each carrying deficiency mutations at different X-linked loci (glucose-6-phosphate dehydrogenase and hypoxanthine-guanine-phosphoribosyltransferase) have been successfully hybridized. The resulting mononucleated hybrid cells have been shown to synthesize both normal gene products, indicating that both X chromosomes are functionally active in the hybrid cells. We believe this is the first reported example of intergenic complementation in fused human diploid cells.