Molecular analysis confirms food source and simultaneous involvement of two distinct but related subgroups of Salmonella typhimurium bacteriophage type 10 in major interprovincial Salmonella outbreak

More than 2,000 confirmed cases of food poisoning occurred in the four Atlantic provinces of Canada and in Ontario during the second and third quarters of 1984. Salmonella typhimurium phage type 10 was identified as the etiologic agent, and cheddar cheese was implicated as the source of infection. Strains isolated from infected humans and from cheese were indistinguishable by biotyping, antibiotic resistance typing, and phage typing. Plasmid analysis confirmed cheese as the source of infection and revealed the presence of two molecular subgroups of bacteriophage type 10. Group I strains carried 57-, 22.3-, and 3.4-kilobase (kb) plasmids; group II strains carried 57-, 4.6-, and 3.4-kb plasmids. Digestion with endonucleases HaeIII, HpaII, and AvaIII indicated that the 3.4-kb plasmids were identical. This outbreak was, therefore, caused by a mixed infection with two distinct but related bacteria. Group I strains are fairly common among Canadian S. typhimurium phage type 10 isolates, whereas group II strains appeared to be unique to this outbreak.     

Molecular analysis confirms food source and simultaneous involvement of two distinct but related subgroups of Salmonella typhimurium bacteriophage type 10 in major interprovincial Salmonella outbreak.

More than 2,000 confirmed cases of food poisoning occurred in the four Atlantic provinces of Canada and in Ontario during the second and third quarters of 1984. Salmonella typhimurium phage type 10 was identified as the etiologic agent, and cheddar cheese was implicated as the source of infection. Strains isolated from infected humans and from cheese were indistinguishable by biotyping, antibiotic resistance typing, and phage typing. Plasmid analysis confirmed cheese as the source of infection and revealed the presence of two molecular subgroups of bacteriophage type 10. Group I strains carried 57-, 22.3-, and 3.4-kilobase (kb) plasmids; group II strains carried 57-, 4.6-, and 3.4-kb plasmids. Digestion with endonucleases HaeIII, HpaII, and AvaIII indicated that the 3.4-kb plasmids were identical. This outbreak was, therefore, caused by a mixed infection with two distinct but related bacteria. Group I strains are fairly common among Canadian S. typhimurium phage type 10 isolates, whereas group II strains appeared to be unique to this outbreak.     

Salmonella serotype typhimurium phage typing. A critical evaluation

Of 12,930 Salmonella serotype typhimurium strains, phage typed during 1985-1988, 45.68% were "nontypable" by Anderson's set; the percent of typable strains decreased from 54.17 in 1986 to 30.54 in 1988. Of 90 phage patterns of sensitivity, 22 were currently encountered. Phage types 1, 18 and 104 were most frequent to strain of both human and non-human origin. In food generating S. typhimurium outbreaks, phage types 1 and 36 were prevalent. Except lysotypes 198 and 95, isolated from "single cases" in man only, all other phage types were common in man and animals, too. Introducing other typing methods to serotype typhimurium "nontypable" strains (by Anderson's set) was considered necessary for epidemiological purposes.     

一起沙门菌引起的食源性疾病爆发的溯源分析

目的:对一起沙门菌引起的食源性疾病爆发进行溯源分析。方法:采用GB4789法对采集的样品进行分离及鉴定,采用16S r RNA基因分型方法及PFGE分型方法对分离的菌株进行分子生物学分析,并对爆发进行溯源分析。结果:生化及血清学结果表明,该起爆发分离的菌型为伦敦沙门氏菌。16S r RNA基因分型表明爆发所分离的菌株均为肠道沙门菌肠道亚种,菌株12 sam与其他4个菌株分子发育距离较远,均为16S r RNA基因分型的TYPE1-11型;PFGE分型结果表明菌株10 sam、16 sam、27 sam及29sam的PFGE带型相似度为100%,菌株12sam跟其他菌株相似率为96%。结论:GB4789法结果表明该起爆发是由伦敦沙门氏菌引起的,16S r RNA基因分型及PFGE分型方法的结果均表明该起食源性疾病来源一致。     

The value of pyrolysis mass spectrometry to investigate nosocomial outbreaks caused by Serratia marcescens

Simultaneous outbreaks of S. marcescens infection going on in the Neonatal Intensive Care Unit and the Surgical Department of the same hospital were investigated by pyrolysis mass spectrometry (PyMS). The PyMS analysis of the strains clearly demonstrated that the two outbreaks were caused by different strains. The 14 S. marcescens isolates from the first outbreak were closely related, with the exception of one environmental isolate, which did not harbour the ESBL plasmid, which was present in all other isolates. However, the phage type of all 14 isolates was the same. Among the 9 S. marcescens isolates from the second outbreak, PyMS clearly distinguished 3 that exhibited gentamicin resistance from the remaining 6 gentamicin-susceptible isolates. Phage typing was unhelpful in this case, as none of the isolates were typable. The PyMS typing of nosocomial outbreak strains can reach the level of discrimination approaching that achieved by molecular genetic analysis.     

Large outbreak of food poisoning caused by Salmonella typhimurium definitive type 49 in mayonnaise.

An investigation was conducted to determine the vehicle of infection of an outbreak of food poisoning in a large metropolitan building early in 1988. A questionnaire was distributed to 700 people who had eaten in the building during the week of the outbreak, and attack rates for specific food were calculated. Food and stool samples, environmental samples, and eggs and environmental swabs from the egg suppliers were examined microbiologically. Altogether 474 questionnaires were returned, 120 people reporting gastrointestinal illness. The illness was significantly associated with foods containing mayonnaise. Salmonella typhimurium definitive type 49 was isolated from 76 of the 84 stool samples containing salmonella and from five of the eight samples taken from the chicken house of the main egg supplier. Mayonnaise was probably the vehicle of infection, which was caused by S typhimurium definitive type 49.     

Multiphasic approach reveals genetic diversity of environmental and patient isolates of Mycobacterium mucogenicum and Mycobacterium phocaicum associated with an outbreak of bacteremias at a Texas hospital

Between March and May 2006, a Texas hospital identified five Mycobacterium mucogenicum bloodstream infections among hospitalized oncology patients using fluorescence high-performance liquid chromatography analysis of mycolic acids. Isolates from blood cultures were compared to 16 isolates from environmental sites or water associated with this ward. These isolates were further characterized by hsp65, 16S rRNA, and rpoB gene sequencing, hsp65 PCR restriction analysis, and molecular typing methods, including repetitive element PCR, random amplified polymorphic DNA PCR, and pulsed-field gel electrophoresis (PFGE) of large restriction fragments. Three of five patient isolates were confirmed as M. mucogenicum and were in a single cluster as determined by all identification and typing methods. The remaining two patient isolates were identified as different strains of Mycobacterium phocaicum by rpoB sequence analysis. One of these matched an environmental isolate from a swab of a hand shower in the patient's room, while none of the clinical isolates of M. mucogenicum matched environmental strains. Among the other 15 environmental isolates, 11 were identified as M. mucogenicum and 4 as M. phocaicum strains, all of which were unrelated by typing methods. Although the 16S rRNA gene sequences matched for all 14 M. mucogenicum isolates, there were two each of the hsp65 and rpoB sequevars, seven PCR typing patterns, and 12 PFGE patterns. Among the seven M. phocaicum isolates were three 16S rRNA sequevars, two hsp65 sequevars, two rpoB sequevars, six PCR typing patterns, and six PFGE patterns. This outbreak represents the first case of catheter-associated bacteremia caused by M. phocaicum and the first report of clinical isolates from a U.S. hospital. The investigation highlights important differences in the available typing methods for mycobacteria and demonstrates the genetic diversity of these organisms even within narrow confines of time and space.     

The elimination of Salmonella typhimurium in sewage sludge by aerobic mesophilic stabilization and lime hydrated stabilization

This study observed the effects of two methods, aerobic mesophilic stabilization and lime hydrated stabilization of sewage sludge upon the survival of Salmonella typhimurium. Raw (primary) sludges from the mechanical biological municipal sewage treatment plant were used. Aerobic stabilization and lime hydrated stabilization were carried out in a laboratory fermentor. Aerobic stabilization was carried out in the mesophilic temperature range (from 25.70+/-0.40 to 37.82+/-1.38 degrees C). Lime hydrated was used at an amount of 10 kg/m(3) for the stabilization. Sludge samples were inoculated with a broth culture of S. typhimurium. Quantitative and qualitative examinations of the presence of S. typhimurium were carried out. Aerobic mesophilic stabilization caused elimination S. typhimurium within 48 h. The T(90) value of S. typhimurium was 6.66+/-0.20 h. During the lime hydrated stabilization pH values significantly increased from 5.66+/-0.07 to 12.12+/-0.02 (P<0.01). S. typhimurium was inactivated within 1h and the T(90) value was 0.19+/-0.01 h. Our study confirmed that the treatment of sewage sludge with lime hydrated was significantly more effective than the aerobic mesophilic stabilization, (P<0.01).     

Genotypes of multidrug-resistant Salmonella enterica serotype typhimurium from two regions of Kenya

A combination of phage typing and pulsed-field gel electrophoresis of Xbal-digested chromosomal DNA has been used to study the epidemiological relationships of multidrug-resistant Salmonella enterica serotype typhimurium from Nairobi (64 isolates) and Kilifi (40 isolates) collected over the period 1994-1997. Isolates from Nairobi belonged to 11 definitive phage types (DTs) encompassing eight different PFGE patterns. In contrast, isolates from Kilifi were mainly DT 56 (60%) and all fell into a single PFGE pattern. The remaining isolates did not conform to a recognisable phage type. We conclude that multidrug-resistant S. typhimurium infections from Nairobi were caused by multiple strains while those from Kilifi were likely to be from a microepidemic caused by a single clone.     

Inter-strain comparison by pyrolysis mass spectrometry of Staphylococcus aureus isolates associated with nosocomial infection

Pyrolysis mass spectrometry was used to characterise Staphylococcus aureus isolates from an outbreak of post-operative wound infections on a mixed surgical ward. The PyMS results were compared with those of phage typing. Both suggested a single strain of S. aureus, of phage type 3C, 55,71, was responsible for six of the 13 wound infections. PyMS differentiated an isolate from a member of staff of similar phage type to the epidemic strain, which had previously been considered to be the point source for the outbreak. PyMS is a rapid and inexpensive technique for investigating nosocomial outbreaks, including those caused by S. aureus and, in this instance, was more discriminatory than phage typing.     

Clonal relations among Salmonella enteritidis phage type 3 outbreak isolates traced by DNA fingerprinting.

Isolates of Salmonella enteritidis PT3, a rare phage type, were recovered from patients and strains were isolated from an outbreak of gastroenteritis that occurred during the summer of 1997 in North-East Sardinia, Italy. To investigate possible clonal involvement in the outbreak and to evaluate the capacity to discriminate among S. enteritidis PT3 strains, a number of molecular typing methods including ribotyping with a mixture of PstI and SphI (PS-ribotyping), PFGE with endonuclease XbaI and RAPD typing with four arbitrary primers was used. The typical XbaI endonuclease generated PFGE pattern also explained the prevalence of highly clonal S. enteritidis PT3 strains in the outbreak and adjacent areas. RAPD fingerprinting with primers OPA 4, OPB 15, OPB17 and P1254 exhibited a single but unique RAPD profile among the outbreak strains from various sources that differed significantly from control strains. The results of this study showed that when an appropriately chosen set of primers is employed, RAPD fingerprinting can be used as an alternative, rapid, highly reproducible technique for tracing the clonal relations of S. enteritidis PT3, and can be more discriminatory than PFGE. Furthermore, this study revealed the possibility of PT3 causing outbreak.     

Continued Surveillance of Serratia marcescens Infections by Bacteriocin Typing: Investigation of Two Outbreaks of Cross-Infection in an Intensive Care Unit

During an 8.5-month period, 198 additional isolates of Serratia marcescens were typed by bacteriocin sensitivity; 154 isolates were typable and were categorized according to our current system of 54 provisional bacteriocin sensitivity patterns. Two outbreaks of nosocomial infection due to S. marcescens occurred in our intensive care unit, involving two and five patients, respectively. The latter outbreak was caused by a strain of S. marcescens which was not sensitive to any of the 10 bacteriocins normally used. Therefore we developed a supplementary procedure based on bacteriocin production rather than bacteriocin sensitivity. Bacteriocin production was induced with mitomycin C, and the crude lysates were applied to 15 provisional bacteriocin indicator strains. The reverse typing procedure was necessary to determine the spread and ultimate subsidence of this particular outbreak of cross-infection.     

Interference with propagation of typing bacteriophages by extrachromosomal elements in Salmonella typhimurium: bacteriophage type 505.

Samonella typhimurium bacteriophage type 505 is the most frequently encountered phage type in the Netherlands and its neighboring countries. Phage type 505 was analyzed with regard o the interference with propagation of the typing phages by the prophages and plasmids, present in the type strain S. typhimurium 505...     

Application of pulsed-field gel electrophoresis to laboratory surveillance of small community outbreaks of Salmonella enterica serovar Typhimurium

Infectious diarrhea syndrome is an important cause of human morbidity around the world, and Salmonella genus remains one of the most prevalent etiology. Salmonella enterica serovar Typhimurium outbreak-associated isolates received by the Laboratory for Enteric Pathogens from N.I.R.D.M.I. "Cantacuzino" for confirmation and typing were analyzed by genomic pulsed-field gel electrophoresis (PFGE) and phage susceptibility testing to establish their relatedness. Both typing methods proved to have similar discriminatory power. The isolates originating from the same outbreak belonged to the same phage type and showed indistinguishable PFGE profiles. The molecular characterization of autochthonal Salmonella enterica Typhimurium outbreak human isolates provided laboratory evidence that epidemiologically related isolates collected from community outbreaks of disease were also genetically related. In order to improve the national and international surveillance of major foodborne pathogens the reference laboratory centers are required to establish and maintain the capacity to perform a wide range of both phenotypic and genotypic methods to support outbreak investigations.     

Ribosomal ribonucleic acid isolated from Salmonella typhimurium: absence of the intact 23S species.

Ribonucleic acid (RNA) isolated by four distinct methods and from a variety of Salmonella typhimurium strains lacked intact 23S ribosomal RNA (rRNA). On sucrose gradients which minimize aggregation, the vast majority of S. typhimurium rRNA sedimented as a 16S peak with a 14S shoulder. RNA from this region of the gradient was resolved into three discrete bands by electrophoresis in formamide. Two very minor S. typhimurium RNA peaks were resolved at 21S and 10S on sucrose gradients, and each peak formed discrete bands in electrophoresis. It is concluded that if S. typhimurium does possess an intact 23S rRNA species, this species is extremely "labile." The absence of isolatable S. typhimurium 23S rRNA possibly reflected in vivo processing of the rRNA before isolation. Under certain conditions, S. typhimurium rRNA formed discrete aggregates which sedimented similarly to intact Escherichia coli 23S rRNA.     

A multiple typing approach is recommended for the laboratory-based surveillance of salmonellosis in Romania

In Romania, Salmonella enterica serovar Typhimurium isolates are currently typed by antimicrobial resistance profiles and phage typing, as part of the national laboratory-based surveillance system of human enteric infections. The aim of the present study was to assess the added value of complementing this approach with molecular fingerprinting, namely pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeats analysis (MLVA). Thirty-six S. Typhimurium isolates received by the Reference Center for Human Salmonella Infections for confirmation and typing from the Microbiology Departments of three Public Health Authorities, were selected for this study. Phage typing revealed that 14 isolates (39%) were nontypeable (NT). Twenty-two isolates were assigned to 5 phage types: DT193 (11 isolates), U302 (7 isolates), DT116 (2 isolates), DT41 (1 isolate) and DT86 (1 isolate). Antimicrobial susceptibility testing showed that all the NT and DT116 isolates were multidrug resistant and extended-spectrum betalactamase producers. All the examined isolates were typeable when using the molecular approach. Both methods gave conclusive and comparable results, documenting the genetic relatedness and discriminating the outbreak isolates from sporadic cases. We conclude that in order to improve outbreak investigation and surveillance of salmonellosis in Romania, the current routine typing of Salmonella isolates should be complemented with at least one of these DNA fingerprinting methods.     

International epidemiological and microbiological study of outbreak of Salmonella agona infection from a ready to eat savoury snack--II: Israel.

OBJECTIVES: To explain an increase in the incidence of salmonellosis caused by Salmonella agona in Israel between October 1994 and January 1995 in the light of an outbreak of S agona phage type 15 infection in England and Wales caused by consumption of a ready to eat savoury snack produced in Israel. DESIGN: Epidemiology of S agona in 1994-5 was analysed and two consecutive, case-control studies of 32 and 26 case-control pairs were performed. Phage typing and molecular methods were used to characterise strains of S agona isolated from cases and samples of the snack in Israel and England and Wales. RESULTS: The increase in the incidence of S agona between October 1994 and January 1995 was countrywide. Cases of infection with group B salmonella increased from 60% to 80% in children under 5 years old. In both case-control studies, cases consumed more of the snack than did controls (4.25 v 2.94 packets per week in the first study (P = 0.086) and 4.04 v 2.37 packets per week in the second study (P = 0.034)). When the two studies were combined there was a significant dose-response relation for the number of packets consumed weekly. Compared with consumption of less than two packets, the odds ratio was 1.43 for between two and six packets and 3.37 for seven or more packets (chi 2 for trend = 5.27, P = 0.02) S agona phage type 15 was isolated from a packet of the snack sold in Israel, and the strain was identical with those isolated from packets and cases in Israel and England and Wales. CONCLUSIONS: This outbreak of S agona was caused by the contamination of a snack produced in Israel. Even under modern operating conditions, large, widespread international outbreaks of foodborne disease can occur. The success of this investigation resulted from excellent international collaboration between public health authorities.     

Conventional and molecular approaches to isolates of Salmonella hadar from sporadic and epidemic cases

In September 1994 an outbreak of gastroenteritis occurred in 437 people who had consumed lunch in the canteen of a factory in Central Italy. Salmonella sp. was isolated from stools of 99 patients and in 73 of them Salmonella hadar was identified. This is the first outbreak caused by this serotype described in Italy. In order to examine the genotypic basis of the epidemic strains, molecular typing was applied to sporadic strains isolated before and after the outbreak episode. For this purpose phage type, resistance to antibiotics, DNA plasmid profile and sites of insertion of the mobile element of IS200 were determined. The epidemic strains were genetically distinct from the non-epidemic isolates; they were shown to be phage type 26, harbouring four small plasmids, were resistant to nalidixic acid and showed a unique characteristic IS200 fingerprint. The typing methods used in this study allowed the identification and discrimination of the outbreak strains from related isolates. They can thus be considered as a tool for epidemiological purposes. In addition we should point out the emerging resistance to nalidixic acid, largely used in veterinary medicine, in Salm . hadar .     

Outbreak of coagulase negative staphylococcus highly resistant to ciprofloxacin in a leukaemia unit.

OBJECTIVE--To define an outbreak of bacteraemia due to coagulase negative staphylococci highly resistant to ciprofloxacin in a leukaemia unit, investigate the source and mode of spread of the outbreak strain, and assess control measures. DESIGN--The outbreak strain was characterised by five different typing methods. Surveillance of patients, staff, and environment was carried out during the outbreak and five months after control measures were introduced. SETTING--A unit with 10 beds for adults with leukaemia and patients receiving bone marrow transplants. The outbreak occurred during a trial of ciprofloxacin for empirical treatment of neutropenic fevers. INTERVENTIONS--Ciprofloxacin was withdrawn from use in the unit and daily bathing with chlorhexidine gluconate solution started. Main outcome measure--The absence of bacteraemia due to the outbreak strain for five months after control measures. RESULTS--During the study 49 patients developed 21 episodes of bacteraemia due to the outbreak strain, which was ciprofloxacin resistant (minimum inhibitory concentration greater than or equal to 128 mg/l), susceptible to phage 155 A9C, and SII biotype and had characteristic immunoblot and DNA fingerprint features. There was a high amount of colonisation of patients but not staff with this strain, which was also wide spread in the environment. The control measures led to rapid resolution of the outbreak and disappearance of the strain from the unit. CONCLUSIONS--In areas where coagulase negative staphylococcal infections are common doctors must be aware of the possibility of cross infection with single strain, and the availability of more discriminatory methods of typing will facilitate the identification and control of such episodes.