How to deal with a hospital outbreak of Legionnaires' disease.

The outbreak of Legionnaires' disease in Glasgow Royal Infirmary is discussed together with the problems such an outbreak poses to the microbiologist. The importance of early diagnosis is stressed. The outbreak was managed by a team drawn up from various disciplines within the hospital. Frank daily reports to the press, together with regular staff meetings with staff representatives helped to allay public anxiety. The subsequent maintenance and monitoring of the wet cooling tower required for the hospital ventilation system have resulted in considerable additional work for the microbiology department but especially for the hospital engineers.

Rapid diagnosis of an outbreak of Legionnaires' disease at Glasgow Royal Infirmary.

In the last three months of 1985 there was an outbreak of legionnaires' disease at Glasgow Royal Infirmary affecting 15 patients and one surgeon; five patients died. Legionnaires' disease was first suspected when a second case of severe nosocomial pneumonia occurred in a high dependency unit. The application of the direct fluorescent antibody test to specimens obtained at bronchoscopy was responsible for the rapid diagnosis of legionnaires' disease, which led to the prescription of appropriate antibiotic treatment and the shutting down of the contaminated cooling tower, thereby containing the outbreak. It also led to a search for further cases. It is suggested that these diagnostic techniques should be included in the investigation of affected patients in an outbreak of pneumonia.

Restriction enzyme fingerprinting of trimethoprim resistance plasmids.

Restriction enzyme fingerprinting was applied to 72 transferable trimethoprim resistance plasmids to examine aspects of their epidemiology and molecular relatedness. These plasmids had previously been divided into 25 groups according to differences in mol. wts and in antimicrobial resistance determinants. Restriction enzyme fingerprinting allowed the plasmids to be further divided into 44 different groups. The groups based on molecular weight and resistance patterns often, but not invariably, corresponded with those based on restriction enzyme fingerprints. Some plasmids with the same mol. wt and resistance pattern had different digest fingerprints and conversely, although more rarely, plasmids which differed in molecular weight by as much as 10 MDa or in resistance pattern by one resistance marker, had indistinguishable fingerprints. The plasmids were initially divided into three broad categories according to which restriction enzymes gave fingerprints of 6-20 fragments. These categories differed in the molecular weights of the plasmids contained, the numbers of resistance markers, and the proportions of the plasmids which carried transposon Tn7. Some plasmids were more widespread and persistent than others with the same mol. wt and resistance pattern but with a different restriction enzyme fingerprint. Thus, application of this technique has shown the trimethoprim resistance plasmids studied to be more diverse than was indicated by determination of mol. wt and resistance pattern, and has indicated changes in the plasmid pool over the 3 years during which they were collected.

A comparison of the distribution of pneumococcal types in systemic disease and the upper respiratory tract in adults and children.

The serotype distribution of 874 strains of Streptococcus pneumoniae was determined in relation to patients' age and to frequency of isolation from systemic disease. Types 14 and 18, in pre-school children, and types 1, 4, 7, 8 and 12 in patients over 5 years of age were significantly associated with systemic disease whereas type 23 in pre-school children, and type 6 in older patients was associated with upper respiratory tract carriage. No significant difference was found in the incidence of other types in systemic disease compared to upper respiratory tract carriage. Fifteen diagnostic pneumococcal antisera (to types 1, 3, 4, 6, 7, 8, 9, 11, 12, 14, 17, 18, 19, 22 and 23) sufficed for typing 87% of strains.

Outbreak of Legionnaires' disease in Glasgow Royal Infirmary: microbiological aspects.

The bacteriological investigation of an outbreak of Legionnaires' disease in Glasgow Royal Infirmary affecting 16 patients is described. Most of the patients had been treated in high-dependency areas on two floors of the hospital supplied by the same two air-conditioned ventilation systems. The source of infection was traced to contamination of a cooling tower from which a plume of spray discharged into the intake vents of the two ventilation systems. Rubber grommets within the cooling tower probably provided a nidus of infection there. The control and management of the outbreak are discussed: a policy of frankness about the course and progress of the investigations was adopted and helped to allay anxiety on the part of both staff and media.

Distribution and genetic location of Tn7 in trimethoprim-resistant Escherichia coli.

A series of 178 strains of Escherichia coli, highly resistant to trimethoprim, isolated from hospital patients and patients in the community between 1979 and 1983, was examined for the presence of Tn7 on a plasmid or on the chromosome only, by transposition to RP4 and restriction endonuclease digestion with Hind III. Of the isolates, 57% carried Tn7. Comparison of hospital isolates from 1979 to 1980 and 1982 showed that although the proportions that carried Tn7 were similar (63% and 57%) there had been a significant change in the genetic location of the transposon. The proportion of plasmid-mediated Tn7 had fallen from 62% to 30% with a corresponding rise in Tn7 located exclusively on the chromosome from 38% to 70%. This change may be the result of continuing transposition of Tn7 from plasmids to the bacterial chromosome followed by plasmid loss. The consequent reduction in the mobility of trimethoprim-resistance genes may in turn lead to changes in the incidence of resistance.

Trimethoprim resistance in urinary coliforms from patients in the community: plasmids and R-transfer.

A study of urinary coliform isolates, from patients attending their general practitioner, between 1981 and 1983 showed that 10.2% were resistant to trimethoprim. Forty-four trimethoprim-resistant Escherichia coli were collected for further study and the results compared with those obtained from hospital isolates collected during 1979-80 and 1982. Thirty-two (73%) were highly resistant to trimethoprim (MIC greater than 1024 mg/l) a similar proportion to that in the hospital isolates. The highly resistant isolates bore a close resemblance to the hospital isolates in that 66% carried multiple plasmids, 97% were multiply drug-resistant and all were highly resistant to sulphamethoxazole. The frequency of antimicrobial resistance transfer (59%) was similar to that from the hospital isolates. More than half of the trimethoprim resistance plasmids characterized were indistinguishable in terms of mol.wt and resistance pattern from those found in isolates from the hospital collections. Our results suggest that, whereas trimethoprim-resistant E. coli are less commonly isolated from patients in the community than in hospitals, many of the community isolates may have originated from a hospital source.

Antimicrobial resistance and the ecology of Escherichia coli plasmids.

Four hundred and seven clinical isolates of Escherichia coli were examined for the presence of plasmids. These isolates comprised 189 which were collected irrespective of antimicrobial resistance (VP) and 218 which were collected on the basis of high-level trimethoprim resistance (TPR). The VP isolates were divided into drug sensitive (VPS) and drug-resistant (VPR) subpopulations. Plasmids were detected in 88% of VP isolates (81% of VPS and 94% of VPR) and 98% of TPR isolates. The distribution of plasmids in both groups and subpopulations was very similar. However, there were small but statistically significant differences between the plasmid distributions. These showed that more isolates in the resistant groups harboured plasmids than in the sensitive subpopulation (VPS) and that the number of plasmids carried by resistant isolates was greater. Multiple drug resistance was significantly more common among TPR isolates than the VPR subpopulation and this was paralleled by increased numbers of plasmids. Fifty-eight per cent of VPR and 57% of TPR isolates transferred antimicrobial resistance and plasmids to E. coli K12. Of the R+ isolates, 60% carried small plasmids (MW less than 20Md) and 52% of these co-transferred with R-plasmids. These results are discussed.

Distribution and transferability of plasmids in trimethoprim-resistant urinary strains of Escherichia coli: a comparative study of hospital isolates.

Trimethoprim-resistant urinary isolates of Escherichia coli, collected in 1982, were studied and the results compared with those obtained for isolates collected during the period 1979-1980. Ninety-eight (81%; a 10% increase) were resistant to trimethoprim 1024 micrograms/ml and 93 (95%) were also resistant to sulphamethoxazole 1024 micrograms/ml. The frequency distributions of plasmids were similar in both collections although there was a significant increase in the number of small plasmids (mol. wt less than or equal to 20 X 10(6] in the 1982 collection. Transfer of resistance was associated with isolates that carried larger numbers of plasmids. A significantly smaller proportion of isolates in this, than in the earlier series, transferred trimethoprim resistance to E. coli K12 suggesting continued transposition of trimethoprim resistance on to the bacterial chromosome. Fifteen different trimethoprim resistance plasmids were identified, of which five, including that found most frequently, were common to both collections. Plasmids which transferred trimethoprim resistance without sulphonamide resistance were more common in the 1982 series. Plasmids which transferred linked trimethoprim and streptomycin resistances, in particular those not carrying other resistance markers, were less common in the 1982 series of isolates. Although the majority of E. coli isolates highly resistant to trimethoprim remained resistant to sulphonamide, our results suggested changes in the genetic location and linkage of the resistance markers.

Distribution and transferability of plasmids in trimethoprim resistant urinary Escherichia coli.

Urinary isolates of Escherichia coli that were resistant to trimethoprim were collected in Glasgow Royal Infirmary during 1979 and 1980. Eighty-eight were resistant to trimethoprim 1024 micrograms/ml and 80 (92%) were also resistant to sulphamethoxazole 1024 micrograms/ml; 73% were multiresistant. Plasmids were detected in 98% of strains and 60% carried two or more. Half of the isolates transferred trimethoprim resistance to E. coli K12 and 70% of these cotransferred resistance to sulphonamide although these markers were often not linked. Trimethoprim resistance was carried on 12 different plasmids, four of which also conferred sulphonamide resistance. All except two carried streptomycin resistance which suggests that Tn7 was probably present. The results are discussed in relation to current prescribing policy.

Physical mapping of temperature-sensitive mutations of herpes simplex virus type 2 by marker rescue.

The physical mapping of six ts mutations of herpes simplex virus type 2 (HSV-2) is presented. The results were obtained from 14 separate intratypic marker rescue experiments and the analysis of 20 HSV-1/HSV-2 intertypic recombinants. The order of these mutations on the physical map of HSV-2 is unambiguous and correlates almost exactly with the previously published genetic map of Timbury & Calder (1976). One of the mutants studied (HSV-2 ts12) has apparently two distinct conditionally lethal ts mutations, one in the long and the other in the short region of the HSV genome.

Genetic retrieval of viral genome sequences from herpes simplex virus transformed cells.

Oncogenic transformation of cultured cells by inactivated herpes simplex virus (HSV) types 1 and 2 has been demonstrated. Expression of HSV information in these transformed cells has been shown by immunofluorescence studies, detection of HSV neutralizing antibody in sera from tumour-bearing animals and by hybridization of HSV-specific RNA. Molecular hybridization studies of DNA from HSV-2 transformed hamster cells have detected up to 40% of the HSV genome present in several copies. Complementation of three HSV-2 temperature-sensitive mutants when superinfecting the RE1 rat embryo cell line (transformed by the HSV-2 temperature-sensitive mutant ts1) suggests that resident viral genes can be expressed. Brown et al. used a similar approach to detect HSV information latent in human ganglia. We report here retrieval of intertypic HSV recombinants from HSV transformed cells after superinfection with ts mutants of the alternative serotype of HSV. Restriction enzyme analysis which clearly differentiates between HSV-1 and HSV-2 DNA has demonstrated the isolation of recombinants spanning the genome and of virus indistinguishable from the original transforming virus.

Complementation with ts mutants of herpes simplex virus.

Tests to detect complementation between ts mutants of herpes simplex virus types 1 and 2 infectious centre and yield of virus assay were investigated. Progeny analysis of both intratypic and intertypic complementation showed a considerable proportion of recombinant or ts+ virus in the progeny; this was more marked in the infectious centre tests. Virus of intermediate temperature-sensitivity was produced in intratypic as well as intertypic complementation. Reduction in the input multiplicity of one of the two mutants in the test to extremely low levels did not prevent complementation, suggesting that non-infectious particles probably contribute to complementation. Demonstration of virus DNA synthesis in mixedly-infected cells at the non-permissive temperature was used to detect complementation between DNA-negative mutants.

Mutant of herpes simplex virus type 2 with temperature-sensitive lesions affecting virion thermostability and DNase activity: identification of the lethal mutation and physical mapping of the nuc-lesion.

We had previously shown that a temperature-sensitive (ts) mutant of herpes simplex virus type 2 strain HG52, ts13, induced a heat-labile DNase activity in infected cells (B. Francke, H. Moss, M. C. Timbury, and J. Hay, J. Virol. 26:209-213, 1978). Earlier work indicated that the mutant also possessed temperature-sensitive infectivity (I. W. Halliburton and M. C. Timbury, J. Gen. Virol. 30:207-221, 1976). In this study temperature-stable revertants of ts13 have been isolated; examination of them revealed that ts13 is a double mutant, with genetically distinct temperature-sensitive lesions affecting nuclease activity and particle stability. The lethal mutation, in the cell system studied, is the latter. Revertants, which all maintain the nuclease lesion, grew well at a high temperature. Physical mapping of the nuclease lesion placed it between 0.12 and 0.21 (fractional length) on the virus genome, quite distant from the lethal mutation at 0.64 to 0.70.