The alkaline phosphatase test is used as an indicator of adequate pasteurisation of milk and cream. A proprietary fluorimetric technique (Fluorophos) is a sensitive and quantitative method for the determination of alkaline phosphatase (ALP) activity in milk products. Currently, adequate pasteurisation of milk products is regarded as confirmed in samples that contain a residual bovine ALP activity of < or =500 mU/litre. This is equivalent to the statutory acceptable level of 4ug phenol/ml required by the EC analytical method. The purpose of the present study was to assess the effectiveness of pasteurisation of milk and cream produced by on-farm dairies. In a longitudinal study over a four-year period, 4,999 samples of milk and cream were collected from 130 on-farm dairies and from two large commercial dairies in NW England for comparison. Bovine ALP activity of >500 mU/litre was deemed as a failure and was found in 3.5% of whole milk, 2.4% semiskimmed milk, 5.0% of skimmed milk, and 39% of cream samples from on-farm dairies. Bovine ALP activity of >100 and <500 mU/litre was found in 18.4% of whole milk, 9.3% of semi-skimmed milk, 13.2% skimmed milk and 44.5% of cream samples from on-farm dairies. Results with skimmed milk samples showed significantly lower bovine ALP activity than whole milk. All 409 milk and cream samples from two large commercial dairies passed the fluorimetric test at less than 500 mU/litre of bovine ALP, and 99% of these milk and cream samples had bovine ALP activity of less than 100 mU/litre. The presence of residual bovine phosphatase indicates a failure and may be due to either inadequate pasteurisation or post pasteurisation contamination with raw milk. Residual bovine phosphatase was demonstrated in 108/114 (94.7%) of milk samples with a bovine ALP activity greater than 500 mU/litre, i.e. true failures. Of more concern is that residual bovine phosphatase was found in 395/401 (98.5%) of samples that gave bovine ALP activity greater than 100 mU/litre but equal to or less than 500 mU/litre. Residual bovine phosphatase was demonstrated in 37/108 (30.2%) of cream samples with bovine ALP activity greater than 500 mU/litre. Presence of reactivated bovine phosphatase is not an indication of a failure but can mask the presence of residual bovine phosphatase. Reactivated bovine phosphatase was found in 74/106 (69.8%) of cream samples. Our results confirm that the more sensitive fluorimetric method is suitable for testing pasteurised whole milk and semiskimmed milk, but for statutory purposes the acceptable level of residual bovine phosphatase should be <100 mU/litre. Our findings have highlighted a potential problem when testing skimmed milk and cream samples from on-farm dairies. To ensure public safety we need more stringent standards for the ALP test and new methods that will accurately confirm that pasteurisation of these products has been achieved.

AIMS:The aim of this study was to identify and subtype a large collection of isolates of Campylobacter spp. to quantify diversity among strains causing human disease from geographically diverse sources in the United Kingdom.METHODS AND RESULTS:Isolates were characterized by the Penner serotyping scheme, Preston phage typing and biotyping methods. The diversity index calculated from the combined results of all three methods was 0.997 and indicated that isolates from sporadic cases of infection are very diverse. Strong associations between common phagetypes (PG52, PG121 and PG55) and the three most common serotypes (HS1, HS2 and HS4) found in the study were evident.CONCLUSIONS:Strains of C. jejuni causing human infections in the United Kingdom are very phenotypically diverse. Individual strains characterized by serotype, phagetype and biotype were detected throughout the 7-month study period and from geographically distinct sources, indicating an unrecognized outbreak or other epidemiologically significant source of human infection.SIGNIFICANCE AND IMPACT OF THE STUDY:The low frequency incidence of most C. jejuni strains should enable easy recognition of outbreaks by strain type surveillance at local, regional and national level in the United Kingdom. The characterization of common strain profiles in this study by simple phenotypic methods could provide the basis for strain specific epidemiological studies for reservoirs of infection and transmission routes for human infection.

In 1995 Preston Public Health Laboratory introduced an incident logging system intended to improve the investigation of suspected outbreaks of infectious intestinal disease. A unique incident log (Ilog) number assigned and issued to the reporting individual and other interested parties when the laboratory is informed of a potential outbreak is used to identify all associated specimens submitted to the laboratory and is quoted in all communications about the incident. The results are reviewed formally each month. Between January 1995 and December 1998, 349 potential outbreaks of infectious intestinal disease were investigated, 325 of which were considered to be general outbreaks. Small round structured viruses were identified in 45% of these outbreaks, salmonellas in 8%, and no pathogens in 35%. Data from the national surveillance scheme for general outbreaks of infectious intestinal disease included 104 general outbreaks in 1996 and 1997 for the entire North West region, but our laboratory alone reported 184 general outbreaks during that period. The Ilog system is a simple and effective means for reviewing data from outbreaks, and helps to coordinate their investigation.

Dr Bolton's present address is Food Safety Microbiology Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT DURING the past decade, multiresistant Salmonella typhimurium definitive phage type (DT) 104 (MR DT104) which is resistant to ampicillin, chloramphenicol, streptomycin/ spectinomycin, sulphonamides and tetracyclines (ACSSpSuT) has caused numerous infections in food animals and human beings in the UK. Infections have also been reported in many European countries as well as in the USA and Canada (Threlfall 2000). In the UK, human infections with MR DT104 are primarily zoonotic in origin (Threlfall and others 1994). Although there has been a decline in isolations from food animals since 1994, MR DT104 remains the most common salmonella strain in cattle, sheep and pigs in the UK, and is second only to Salmonella enteritidis in poultry (Anon 1999a). In human salmonellosis in England and Wales, MR DT104 has declined from a peak of 4006 isolations in 1996 (Threlfall and others 1999) to 1030 isolations in 1999 (PHLS, unpublished observations). Nevertheless, the organism remains second only to S enteritidis phage type 4 in human beings in England and Wales. Antimicrobial drugs are used under veterinary prescription for therapy and prophylaxis in food-producing animals. In 1997 over 70 per cent of isolates of S typhimurium from livestock were resistant to ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracyclines, and a substantial proportion of the resistant isolates were MR DT104 (Anon 1999b). Recently, particular concern has surrounded the isolation of an increasing proportion of MR DT104 with additional resistance to the first generation quinolone nalidixic acid (Nx) in isolations from food-producing animals (Davies and others 1999). Similarly, there have been reports of decreased susceptibility to the fluoroquinolone antimicrobial ciprofloxacin in isolations of MR DT104 from human beings (Threlfall and others 1998). Ciprofloxacin is the first-line drug for the treatment of invasive salmonellosis in humans. In 1994, 1 per cent of MR DT104 from human beings in England and Wales showed decreased susceptibility to ciprofloxacin, and by 1998 this figure had risen to 16 per cent (Threlfall and others 1999). Although the ciprofloxacin minimum inhibitory concentrations (MICs) are below the breakpoint concentration of 1 mg/litre as recommended by the British Society for Antimicrobial Chemotherapy (Anon 1996), S typhimurium isolates with similar MICs have been associated with treatment failure (Piddock and others 1990). In particular, in a recent outbreak of MR DT104 in Denmark, four of 11 patients did not respond to treatment with ciprofloxacin and there were two deaths (M0lbak and others 1999). The increase in the proportion of isolates of MR DT104 with decreased susceptibility to ciprofloxacin in England and Wales is, therefore, particularly disturbing. Between August 23 and September 6, 1998, a large community outbreak of MR DT104 (86 cases) occurred in northwest Lancashire (Anon 1998), and at least four patients were admitted to hospital. Epidemiological investigations revealed that 79 per cent of cases had consumed milk from a local dairy which received raw milk supplied by two farms. MR DT104 was isolated from the milk filter and failure of on-farm pasteurisation was thought to be the cause of the outbreak. On two occasions, in March and August 1998, S typhimurium, subsequently identified as MR DT104, was isolated from dairy cattle on one of the farms supplying the dairy. Strains from human beings, the dairy cattle and the milk filter were characterised. All were of R-type ACSSpSuTNXCPL (Nx nalidixic acid, CPL ciprofloxacin [L:MIC 0.5 mg/litre]) and possessed a single 90 kilobase (kb) plasmid. When a selection of isolates were typed by pulsed-field gel electrophoresis (PFGE), all gave a single PFGE pattern (XTmi) identical to that predominant in MR DT104 (Ridley and Threlfall 1998, Prager and others 1999). Long PCR (R. A. Walker, unpublished observations) demonstrated that these isolates also possessed the chromosomally-encoded 13 kb gene cluster that confers resistance to ACSSpSuT and is characteristic of MR DT104 (Briggs and Fratamico 1999). To investigate further the clonal nature of the strains, the mechanism of resistance to nalidixic acid and ciprofloxacin was determined in isolates from human beings, dairy cattle and the milk filter. The primary mechanism of resistance to nalidixic acid and ciprofloxacin commonly involves a single nucleotide mutation of the gyrA gene (Piddock and others 1998). Mutations in the gyrA gene were therefore characterised, using a rapid LightCycler assay (Gibson and others 1999). This involved amplifying a region of gyrA by PCR followed by the detection of ciprofloxacin resistance-associated mutations using hybridisation probes and thermal analysis. All the isolates were found to have a GAC-*GGC (aspartate to glycine) substitution at codon 87, subsequently confirmed by DNA sequencing. Ten different base pair substitutions in gyrA have been identified in ciprofloxacin-resistant salmonellas. However, although the GAC-*GGC substitution has been observed in MR DT104 (Ridley and Threlfall 1998), the most common mutation in strains with decreased susceptibility to ciprofloxacin involves a GAC->AAC (aspartate to asparagine) mutation at codon 87 (Ridley and Threlfall 1998, Molbak and others 1999, R. A. Walker, unpublished observations). In this outbreak, identification of the GAC->GGC gyrA mutation at codon 87 provided further confirmation of clonal identity of the strains involved. The emergence of decreased susceptibility to ciprofloxacin in MR DT104 is subsequent to the licensing in the UK, in November 1993, of the related fluoroquinolone enrofloxacin for use in food-producing animals. However, there is considerable controversy over the apparent association between enrofloxacin usage in food animals and the emergence of strains with decreased susceptibility to ciprofloxacin in human beings. Indeed, it has been stated that without evidence of scientific data, such an association cannot be substantiated (Jones 1998). However, this investigation has demonstrated a distinct mutation in the gyrA gene in isolates of MR DT104 from dairy cattle, a milk filter and human beings in a defined outbreak situation. An identical gyrA mutation was also identified in MR DT104 isolated from dairy cattle on one of the farms four months before the outbreak. The fluoroquinolone antimicrobial marbofloxacin was in use on the farm in the months preceding the outbreak (T. Leonard, personal communication). Although it is common for subclinical infection of cattle herds with MR DT104 to persist for several months or years (Davies 1997), it is possible

The photodynamic antibacterial properties of a closely related series of phenothiazinium dyes were tested against several pathogenic strains of Staphylococcus aureus, four of which were methicillin-resistant. Illumination of the photosensitisers at a fluence rate of 1.75 mW cm-2 generally resulted in the enhancement of antibacterial activity in liquid culture and in greater efficacy than the methicillin analogue flucloxacillin. For methylene blue, dimethyl methylene blue and new methylene blue illumination led to increases in bactericidal activity < or = 16-fold, typically 4-fold. In addition dimethyl methylene blue and new methylene blue were active against epidemic strains of methicillin-resistant Staphylococcus aureus at concentrations lower than that of vancomycin (> or = 0.5 microM).