In vitro effects of azithromycin on Salmonella typhi: early inhibition by concentrations less than the MIC and reduction of MIC by alkaline pH and small inocula.

To explain good clinical results of azithromycin in patients with typhoid fever, 10 strains of Salmonella typhi were grown in cation-adjusted Mueller-Hinton broth. MICs of azithromycin were 4-16 mg/L. At a sub-MIC of 2 mg/L, early inhibition of growth was shown at 2, 4 and 8 h of incubation, but at 24 and 48 h growth to turbidity occurred. At 4 mg/L, inhibition occurred up to 8 h, after which growth towards turbidity followed. Elongated curved bacilli formed in broth containing 4 mg/L after 24-48 h. Adjusting the pH of the broth with phosphate-citrate buffer to 7.5 and 8.0 caused reductions in MICs to 0.25-0.5 mg/L. Large inocula of 10(6) cfu/mL resulted in median MICs four- to six-fold greater than with inocula of 10(1)-10(3) cfu/mL. An inoculum of 10 bacteria per mL in broth at pH 7.5 resulted in an MIC of 0.13 mg/L. Clinical benefits in patients may occur because of early inhibition by sub-MIC concentrations of azithromycin, and due to lower MICs at alkaline pH and lower MICs with small inocula that may correspond to the low-grade bacteraemia in typhoid fever.

Differentiation of clinical Helicobacter pullorum isolates from related Helicobacter and Campylobacter species.

BACKGROUND: Helicobacter pullorum, first detected in the liver and intestinal contents of poultry, was defined as a new species in 1994. This organism has since been isolated from humans with gastroenteritis. Phenotypic as well as genotypic methods have been used to identify H. pullorum associated with cases of human disease. MATERIALS AND METHODS: Clinical isolates were submitted for identification to the National Laboratory for Enteric Pathogens by Provincial Public Health Laboratories within Canada. Phenotypic characterization was conducted using a variety of growth and biochemical tests including oxidase, catalase, indoxyl acetate, H2S production in triple sugar iron (TSI) agar, antimicrobial susceptibility testing, and fatty acid analysis. Genotypic identification was performed using a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis of a 1-kb fragment of the Helicobacter 16S rRNA gene. RESULTS: During the last 7 years (1993-1999) a total of 11 isolates of H. pullorum were detected from patients with gastroenteritis for inclusion in this study. Typically, these isolates were oxidase and catalase positive, produced optimal growth at 42 degrees C, and produced H2S in TSI. Of these 11 isolates, 1 showed DNase activity, while another did not produce H2S in TSI, and only 2 showed tolerance to 1% bile. Antimicrobial susceptibility assays indicated that 6 of the 11 strains were resistant to nalidixic acid. The fatty acid profiles of the isolates were similar to each other and provided a distinguishing profile from the other related species. Genetically identical and distinct species-specific restriction fragment-length polymorphism (RFLP) patterns were produced using the restriction enzymes Bsr I and Dde I. CONCLUSION: Phenotypic and genotypic procedures were used to identify H. pullorum. Interspecies phenotypic variability was apparent and supported the use of a polyphasic approach for identification. Similarities to the more prominent human pathogens Campylobacter coli and C. lari were also noted. The use of a combination of phenotypic and, in particular, genotypic markers for H. pullorum should prove valuable both for epidemiological investigations and for the diagnosis of disease related to this emerging human pathogen.

Epidemiologic subtyping of Escherichia coli serogroup O157 strains isolated in Ontario by phage typing and pulsed-field gel electrophoresis.

Phage typing and DNA macrorestriction fragment analysis by pulsed-field gel electrophoresis (PFGE) were evaluated for use in the epidemiological subtyping of Escherichia coli serogroup O157 strains isolated in Ontario, Canada. Among 30 strains isolated from patients with sporadic cases of infection, 22 distinct XbaI macrorestriction patterns were identified and 17 strains exhibited unique PFGE patterns. In contrast, phage typing identified only seven different phage types and 17 strains belonged to the same phage type. A total of 25 phage type-macrorestriction pattern combinations were identified among the strains from patients with sporadic cases of infection. PFGE subtyping differentiated between unrelated strains that exhibited the same phage type, and in one group of strains, phage typing differentiated between strains of the same PFGE subtype. Both typing procedures correctly identified outbreak-related isolates as belonging to the same type in four separate outbreaks. Each outbreak strain was characterized by a distinct macrorestriction pattern, while phage typing subdivided the outbreak strains into only three different types. A small percentage of outbreak-related isolates had PFGE patterns that differed slightly (one or two DNA fragment differences) from that of the outbreak strain. On the other hand, each isolate from the same outbreak belonged to the same phage type as that of the outbreak strain. We conclude that phage typing and PFGE fingerprinting represent complementary procedures for the subtyping of E. coli serogroup O157 and that the combined use of these procedures provides optimal discrimination.

Epidemiologic typing of Salmonella enterica serotype enteritidis in a Canada-wide outbreak of gastroenteritis due to contaminated cheese.

A major Canada-wide outbreak of gastroenteritis due to Salmonella enterica serotype Enteritidis phage type (PT) 8 occurred in 1998, and this was traced to contaminated cheese in a commercial lunch pack product. Phage typing and pulsed-field gel electrophoresis linked the clinical and cheese isolates of serotype Enteritidis but failed to differentiate outbreak from nonoutbreak PT 8 strains. Further differentiation was made by biotyping based on melibiose fermentation.

Multiresistant Salmonella Typhimurium DT104 infections of humans and domestic animals in the Pacific Northwest of the United States.

Salmonella Typhimurium definitive type 104 with chromosomally encoded resistance to five or more antimicrobial drugs (R-type ACSSuT+) has been reported increasingly frequently as the cause of human and animal salmonellosis since 1990. Among animal isolates from the northwestern United States (NWUS), R-type ACSSuT+ Typhimurium isolates increased through the early 1990s to comprise 73% of Typhimurium isolates by 1995, but subsequently decreased to comprise only 30% of isolates during 1998. NWUS S. Typhimurium R-type ACSSuT+ were consistently (99%) phage typed as DT104 or the closely related DTu302. S. Typhimurium isolates from cattle with primary salmonellosis, randomly selected from a national repository, from NWUS were more likely to exhibit R-type ACSSuT+ (19/24, 79%) compared to isolates from other quadrants (17/71, 24%; P < 0.01). Human patients infected with R-type ACSSuT+ resided in postal zip code polygons of above average cattle farm density (P < 0.05), while patients infected with other R-types showed no similar tendency. Furthermore, humans infected with R-type ACSSuT+ Typhimurium were more likely to report direct contact with livestock (P < 0.01) than humans infected with other R-types.

Treatment of typhoid fever with azithromycin versus chloramphenicol in a randomized multicentre trial in India.

To compare the clinical and bacteriological efficacies of azithromycin and chloramphenicol for treatment of typhoid fever, 77 bacteriologically evaluable adults, with blood cultures positive for Salmonella typhi or Salmonella paratyphi A susceptible to their assigned drugs, were entered into a randomized open trial at four hospitals in India. Forty-two patients were randomized to receive azithromycin 500 mg p.o. od for 7 days and 35 to receive chloramphenicol 2-3 g p.o. od in four divided doses for 14 days. Thirty-seven patients (88%) in the azithromycin group responded with clinical cure or improvement within 8 days and 30 patients (86%) in the chloramphenicol group responded with cure or improvement. By day 14 after the start of treatment, all patients treated with azithromycin and all except two of the patients treated with chloramphenicol (94%) were cured or improved. Blood cultures repeated on day 8 after start of therapy showed eradication of organisms in 100% of patients in the azithromycin group and 94% of patients in the chloramphenicol group. By day 14 the eradication rate in the chloramphenicol group had increased to 97%. Stool cultures on days 21 and 35 after start of treatment showed no prolonged faecal carriage of Salmonella spp. in either group. These results indicate that azithromycin given once daily for 7 days was effective therapy for typhoid fever in a region endemic with chloramphenicol-resistant S. typhi infection and was equivalent in effectiveness to chloramphenicol given to patients with chloramphenicol-susceptible infections.

Azithromycin versus ciprofloxacin for treatment of uncomplicated typhoid fever in a randomized trial in Egypt that included patients with multidrug resistance.

To compare clinical and bacteriological efficacies of azithromycin and ciprofloxacin for typhoid fever, 123 adults with fever and signs of uncomplicated typhoid fever were entered into a randomized trial. Cultures of blood were positive for Salmonella typhi in 59 patients and for S. paratyphi A in 3 cases; stool cultures were positive for S. typhi in 11 cases and for S. paratyphi A in 1 case. Multiple-drug resistance (MDR; resistance to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole) was present in isolates of 21 of 64 patients with positive cultures. Of these 64 patients, 36 received 1 g of azithromycin orally once on the first day, followed by 500 mg given orally once daily on the next 6 days; 28 patients received 500 mg of ciprofloxacin orally twice daily for 7 days. Blood cultures were repeated on days 4 and 10 after the start of therapy, and stool cultures were done on days 4, 10, and 28 after the start of therapy. All patients in both groups improved during therapy and were cured. Defervescence (maximum daily temperatures of

Salmonella typhimurium DT104: a virulent and drug-resistant pathogen.

Salmonella typhimurium phage type (PT) or definitive type (DT) 104 is a virulent pathogen for humans and animals, particularly cattle. It has been isolated increasingly from humans and animals in the United Kingdom and several other European countries and, more recently, in the United States and Canada. Humans may acquire the infection from foods of animal origin contaminated with the infective organism. Farm families are particularly at risk of acquiring the infection by contact with infected animals or by drinking unpasteurized milk. The symptoms in cattle are watery to bloody diarrhea, a drop in milk production, pyrexia, anorexia, dehydration and depression. Infection may result in septicemic salmonellosis and, upon necropsy, a fibrinonecrotic enterocolitis may be observed. The infection occurs more commonly in the calving season than at other times. Feedlot cattle and pigs may also be affected. Prolonged carriage and shedding of the pathogen may occur. Symptoms in humans consist of diarrhea, fever, headache, nausea, abdominal pain, vomiting, and, less frequently, blood in the stool. Salmonella typhimurium DT104 strains are commonly resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline.

Combined use of bacteriophage typing and pulsed-field gel electrophoresis in the epidemiological analysis of Japanese isolates of enterohemorrhagic Escherichia coli O157:H7.

A total of 236 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates in Japan were investigated by bacteriophage typing, and the results were compared with those of pulsed-field gel electrophoresis (PFGE). Seven phage types (PTs) were observed in 71 isolates which were derived from 22 outbreaks. All of the isolates from ten outbreaks in the Kinki region (midwestern part of Japan) in July-August 1996 were grouped into the same PFGE type (IIa) and PT 32, while among total isolates, there were such varieties as PFGE type IIa containing five phage types and PT32 containing two PFGE types. These results suggest that the ten outbreaks should be considered to be a single outbreak, and show that the combined use of bacteriophage typing and PFGE enhances reliability in epidemiological surveys.

Isolation and characterization of sorbitol-fermenting Shiga toxin (Verocytotoxin)-producing Escherichia coli O157:H- strains in the Czech Republic.

Two sorbitol-fermenting (SF) Shiga toxin-producing Escherichia coli (STEC) O157:H- strains were isolated from patients with hemolytic-uremic syndrome in the Czech Republic in 1995. Their phenotypic and genotypic characteristics and genomic DNA fingerprints were identical or closely related to those of SF STEC O157:H- strains isolated in Germany in 1988 to 1997. This indicates that the Czech isolates belong to the SF STEC O157 clone which is widespread in Germany. It is the first finding of the clone outside Germany.

Molecular characterization of multiresistant strains of Salmonella typhi from South Asia isolated in Ontario, Canada.

Two hundred and fourteen isolates of Salmonella typhi submitted to our laboratory between 1992 and 1996 were tested for susceptibility to 20 antimicrobial agents. Forty-eight of the 214 isolates (22.4%), recovered from individuals who had travelled in South Asia, were multiresistant. Forty-four of the 48 isolates were resistant to ampicillin, chloramphenicol, tetracycline, streptomycin, sulfamethoxazole, trimethoprim, cotrimoxazole, ticarcillin, and piperacillin; the other four isolates were resistant to four to six agents. Forty-two of the multiresistant isolates belonged to Vi phage type E1, two isolates from the Punjab State belonged to phage type A, another from the Punjab State belonged to phage type E3, one isolate from Pakistan belonged to type M1, and one isolate from India belonged to type J1. Plasmids from 45 of 48 isolates showed a temperature-sensitive mechanism of transfer to Escherichia coli K-12 strains, characteristic of H1 incompatibility group plasmids. The majority of plasmids had an estimated molecular weight of 120 MDa and encoded both citrate utilization and mercury resistance. Plasmids from three isolates had an estimated molecular weight of 112-115 MDa; one of these isolates encoded citrate utilization but not mercury resistance. Analysis of isolates by pulsed-field gel electrophoresis after digestion with XbaI and SpeI indicated that the majority of multiresistant isolates shared a common restriction profile, while four isolates had unique patterns.

Escherichia coli O157:H7 diarrhoea associated with well water and infected cattle on an Ontario farm.

A 16-month old female child living on an Ontario dairy farm was taken to hospital suffering from bloody diarrhoea. Escherichia coli O157:H7 was isolated from her stool. Initial tests of well water samples were negative for E. coli by standard methods but culture of selected coliform colonies on sorbitol-MacConkey agar led to isolation of E. coli O157:H7. E. coli O157:H7 was also isolated from 63% of cattle on the farm. The E. coli O157:H7 isolates from the child, the water and the cattle were phage type 14, produced verotoxins 1 and 2, and were highly related on analysis by pulsed field gel electrophoresis. The child did not have known direct contact with the cattle and did not consume unpasteurized milk. Hydrogeological investigation revealed the design and location of the well would allow manure-contaminated surface water to flow into the well. This investigation demonstrates that cattle farm well water is a potential source of E. coli O157:H7 which may not be identified by standard screening for E. coli in water.

Human salmonellosis associated with exotic pets.

During the period from 1994 to 1996, an increase in the number of laboratory-confirmed cases of human salmonellosis associated with exposure to exotic pets including iguanas, pet turtles, sugar gliders, and hedgehogs was observed in Canada. Pet turtle-associated salmonellosis was recognized as a serious public health problem in the 1960s and 1970s, and in February 1975 legislation banning the importation of turtles into Canada was enacted by Agriculture Canada. Reptile-associated salmonellosis is once again being recognized as a resurgent disease. From 1993 to 1995, there were more than 20,000 laboratory-confirmed human cases of salmonellosis in Canada. The major source of Salmonella infection is food; however, an estimated 3 to 5% of all cases of salmonellosis in humans are associated with exposure to exotic pets. Among the isolates from these patients with salmonellosis, a variety of Salmonella serotypes were also associated with exotic pets and included the following: S. java, S. stanley, S. poona, S. jangwani, S. tilene, S. litchfield, S. manhattan, S. pomona, S. miami, S. rubislaw, S. marina subsp. IV, and S. wassenaar subsp. IV.

Salmonella isolated from humans, animals and other sources in Canada, 1983-92.

A total of 89760 human and 22551 non-human isolates of salmonella were serotyped in Canada during the period 1983-92. There were 2180 reported outbreaks associated with 10065 cases during the 10-year period. The most common salmonella serovars isolated from human and non-human sources were S. typhimurium and S. hadar. The third and fourth most common serovars from human sources were S. enteritidis and S. heidelberg, respectively, and from non-human sources they were S. heidelberg and S. infantis. The number of S. typhimurium isolations from human and non-human sources showed a downward trend over the 10-year period. A total of 222 outbreaks of S. typhimurium associated with 1622 cases occurred. The S. hadar isolations from human and non-human sources reached a peak during the years 1987-90 and declined thereafter. The number of human isolates of S. enteritidis increased until 1985 and fluctuated at a level of 8.3-12.8% of all human isolates thereafter. Seventy-three outbreaks of S. enteritidis infection associated with 568 cases occurred. More than 50% of the S. enteritidis infections in humans were caused by phage type (PT) 8. During the review period, infections caused by PT4 were less common and were almost exclusively found in people who had travelled abroad. The annual isolation rates of S. heidelberg from human and non-human sources increased steadily during the period. Bacteriophage typing of serovars from outbreaks showed that contaminated food products of poultry and bovine origin were common sources of human infection. Salmonella typhi was identified as the cause of 43 small outbreaks affecting 116 persons.

Screening for Salmonella with a murine monoclonal antibody M105 detects both Felix O1 bacteriophage sensitive and resistant Salmonella strains.

Ten Felix O1 (FO1) bacteriophage sensitive Salmonella strains as well as their phage resistant derivates together with 39 strains of FO1-resistant Salmonella were tested for their reactivities with a murine monoclonal antibody, M105, by indirect whole cell and competitive ELISA. All FO1 phage sensitive and 48 of the 49 FO1-resistant Salmonella strains were found to react with M105. The single Salmonella strain not reacting with M105 was a FO1 resistant derivative selected by exposing the sensitive parent strain to the phage. This M105-negative and FO1-resistant strain was also found to be a rough mutant without O-antigens and possibly lacks the terminal LPS core sugars which form the M105 reactive epitope.

Outbreak of Salmonella give in the province of Quebec.

Salmonellosis due to Salmonella Give involving cows, a goat, and an ostrich over a 6-month period was investigated. Cases were found in 4 different regions. Antimicrobial susceptibility patterns, phagetyping, and pulsed-field gel electrophoresis analyses confirmed that all isolates belonged to the same clone. A common source of infection was suspected.