Bala Swaminathan

Epidemiology of human listeriosis.

During the 1980s, investigation of several large epidemics of listeriosis confirmed that transmission of L. monocytogenes in food causes human disease. Progress in laboratory detection and subtyping of the organism has enhanced our ability to compare human and environmental isolates of L. monocytogenes. Transmission by foodborne organisms is now recognized as causing both epidemic and sporadic listeriosis. Continued study of dietary risk factors associated with listeriosis is needed in order to develop dietary recommendations for the expanding population at increased risk of disease. Current research application of new molecular methods to the study of L. monocytogenes may improve the ability to diagnose pregnancy-associated disease and permit the rapid detection and control of L. monocytogenes in the food supply.

An Outbreak of Gastroenteritis and Fever Due to Listeria monocytogenes in Milk

BACKGROUND After an outbreak of gastroenteritis and fever among persons who attended a picnic in Illinois, chocolate milk served at the picnic was found to be contaminated with Listeria monocytogenes. METHODS In investigating this outbreak, we interviewed the people who attended the picnic about what they ate and their symptoms. Surveillance for invasive listeriosis was initiated in the states that receive milk from the implicated dairy. Stool and milk samples were cultured for L. monocytogenes. Serum samples were tested for IgG antibody to listeriolysin O. RESULTS Forty-five persons had symptoms that met the case definition for illness due to L. monocytogenes, and cultures of stool from 11 persons yielded the organism. Illness in the week after the picnic was associated with the consumption of chocolate milk. The most common symptoms were diarrhea (present in 79 percent of the cases) and fever (72 percent). Four persons were hospitalized. The median incubation period for infection was 20 hours (range, 9 to 32), and persons who became ill had elevated levels of antibody to listeriolysin O. Isolates from stool specimens from patients who became ill after the picnic, from sterile sites in three additional patients identified by surveillance, from the implicated chocolate milk, and from a tank drain at the dairy were all serotype 1/2b and were indistinguishable on multilocus enzyme electrophoresis, ribotyping, and DNA macrorestriction analysis. CONCLUSIONS L. monocytogenes is a cause of gastroenteritis with fever, and sporadic cases of invasive listeriosis may be due to unrecognized outbreaks caused by contaminated food.

Variation in virulence among clades of Escherichia coli O157:H7 associated with disease outbreaks

Escherichia coli O157:H7, a toxin-producing food and waterborne bacterial pathogen, has been linked to large outbreaks of gastrointestinal illness for more than two decades. E. coli O157 causes a wide range of clinical illness that varies by outbreak, although factors that contribute to variation in disease severity are poorly understood. Several recent outbreaks involving O157 contamination of fresh produce (e.g., spinach) were associated with more severe disease, as defined by higher hemolytic uremic syndrome and hospitalization frequencies, suggesting that increased virulence has evolved. To test this hypothesis, we developed a system that detects SNPs in 96 loci and applied it to >500 E. coli O157 clinical strains. Phylogenetic analyses identified 39 SNP genotypes that differ at 20% of SNP loci and are separated into nine distinct clades. Differences were observed between clades in the frequency and distribution of Shiga toxin genes and in the type of clinical disease reported. Patients with hemolytic uremic syndrome were significantly more likely to be infected with clade 8 strains, which have increased in frequency over the past 5 years. Genome sequencing of a spinach outbreak strain, a member of clade 8, also revealed substantial genomic differences. These findings suggest that an emergent subpopulation of the clade 8 lineage has acquired critical factors that contribute to more severe disease. The ability to detect and rapidly genotype O157 strains belonging to such lineages is important and will have a significant impact on both disease diagnosis and treatment guidelines.

Rapid Pulsed-Field Gel Electrophoresis Protocol for Subtyping of Campylobacter jejuni

ABSTRACT We developed a rapid pulsed-field gel electrophoresis (PFGE) protocol for subtyping Campylobacter isolates based on the standardized protocols used by PulseNet laboratories for the subtyping of other food-borne bacterial pathogens. Various combinations of buffers, reagents, reaction conditions (e.g., cell suspension concentration, lysis time, lysis temperature, and restriction enzyme concentration), and electrophoretic parameters were evaluated in an effort to devise a protocol that is simple, rapid, and robust. PFGE analysis of Campylobacter isolates can be completed in 24 to 30 h using this protocol, whereas the most widely used current protocols require 3 to 4 days to complete. Comparison of PFGE patterns obtained in six laboratories showed that subtyping results obtained using this protocol are highly reproducible.

Bartonella (Rochalimaea) quintana Bacteremia in Inner-City Patients with Chronic Alcoholism

BACKGROUND Bartonella (Rochalimaea) quintana is a fastidious gram-negative bacterium known to cause trench fever, cutaneous bacillary angiomatosis, and endocarditis. Between January and June 1993 in Seattle, we isolated B. quintana from 34 blood cultures obtained from 10 patients not known to be infected with the human immunodeficiency virus (HIV). METHODS After identifying the isolates as B. quintana by direct immunofluorescence and DNA-hybridization studies, we determined strain hybridization with studies of restriction-fragment-length polymorphisms (RFLPs) of the intergenic spacer (noncoding) region of ribosomal DNA amplified by the polymerase chain reaction (PCR). To characterize the epidemiologic and clinical features of bartonella infections in these patients, we performed a retrospective case-control study using as controls 20 patients with blood cultures obtained at approximately the same time as those obtained from the index patients. RESULTS B. quintana isolates from the 10 patients were indistinguishable by PCR-RFLP typing. All 10 patients had chronic alcoholism, and 8 were homeless (P = 0.001 for both comparisons with controls). The six patients who underwent HIV testing were seronegative. At the time of their initial presentation, seven patients had temperatures of at least 38.5 degrees C. Six patients had three or more blood cultures that were positive for B. quintana, and in four of these patients B. quintana was isolated from blood cultures obtained 10 or more days apart. Subacute endocarditis developed in two patients and required surgical removal of the infected aortic valve in one of them. Nine patients recovered; one died of sepsis from Streptococcus pneumoniae infection. CONCLUSIONS B. quintana is a cause of fever, bacteremia, and endocarditis in HIV-seronegative, homeless, inner-city patients with chronic alcoholism.

Utility of Multilocus Sequence Typing as an Epidemiological Tool for Investigation of Outbreaks of Gastroenteritis Caused by Campylobacter jejuni

ABSTRACT Multilocus sequence typing (MLST) has been proven useful for the study of the global population structure of Campylobacter jejuni; however, its usefulness for the investigation of outbreaks of disease caused by C. jejuni has not been proven. In this study, MLST plus sequencing of the flaA short variable region (SVR) were applied to 47 isolates from 12 outbreaks of C. jejuni infection whose relatedness has been determined previously, and the results were compared to those of serotyping and pulsed-field gel electrophoresis (PFGE). Isolates implicated in an outbreak were indistinguishable by all four subtyping methods, with sporadic isolates being distinguished from outbreak isolates. Two sporadic isolates from one outbreak were resistant to SmaI digestion and therefore nontypeable by PFGE but were differentiated from the outbreak strain by the other methods. PFGE and flaA SVR typing were the most discriminatory methods, with discriminatory indices (DI) of 0.930 and 0.923, respectively. However, an epidemic strain from one outbreak was distinguished from the other outbreak isolates by flaA SVR typing; its flaA allele was different at five nucleotides, suggesting that this change was possibly mediated by recombination. MLST was less discriminatory than PFGE and flaA SVR typing (DI = 0.859), and many of the epidemic strains possessed common sequence types (STs) including ST-8, -21, -22, and -42. However, further discrimination within STs was achieved by flaA SVR typing or PFGE. The results from this study demonstrate that a combined approach of MLST plus flaA SVR typing provides a level of discrimination equivalent to PFGE for outbreak investigations.

Listeria monocytogenes contamination of turkey franks: Evaluation of a production facility

A facility which produced turkey franks that had been microbiologically linked to a case of human listeriosis was evaluated to establish prevalence of contamination and identify potential points for intervention. Listeria monocytogenes was isolated from only two of 41 environmental samples obtained in the plant. Among production line product samples analyzed by the Centers for Disease Control, 0 to 8% of samples from the production stages before the peeler-conveyor belt apparatus were positive for the case strain of L. monocytogenes , whereas 12 of 14 (86%) samples collected from this apparatus were positive (p <0.001). The most probable number (MPN) of L. monocytogenes in finished product purchased from a retail outlet was less than 0.3 per gram; however, the opened package of franks from the case patients refrigerator had an MPN of >1100 per gram. These data suggest that systematic culturing and analysis of products and production facilities may help identify appropriate interventions to reduce L. monocytogenes contamination in food processing plants and contribute to control of L. monocytogenes in ready-to-eat meat products.

Genetic Markers Unique to Listeria monocytogenes Serotype 4b Differentiate Epidemic Clone II (Hot Dog Outbreak Strains) from Other Lineages

ABSTRACT A small number of closely related strains of Listeria monocytogenes serotype 4b, designated epidemic clone I (ECI), have been implicated in numerous outbreaks of food-borne listeriosis described during the past two decades in Europe and North America. In 1998 to 1999, a multistate outbreak traced to contaminated hot dogs involved a different strain type of serotype 4b, with genetic fingerprints rarely encountered before. In spite of the profound economic and public health impact of this outbreak, the implicated bacteria (designated epidemic clone II [ECII]) have remained poorly characterized genetically, and nucleotide sequences specific for these strains have not been reported. Using genome sequence information, PCR, and Southern blots, we identified DNA fragments which appeared to be either absent or markedly divergent in the hot dog outbreak strains but conserved among other serotype 4b strains. PCR with primers derived from these fragments as well as Southern blots with the amplicons as probes readily differentiated ECII from other serotype 4b strains. The serotype 4b-specific region harboring these fragments was adjacent to inlA, which encodes a well-characterized virulence determinant. The findings suggest that ECII strains have undergone divergence in portions of a serotype-specific region that is conserved in other serotype 4b strains. Although the mechanisms that drive this divergence remain to be identified, DNA-based tools from this region can facilitate the detection and further characterization of strains belonging to this lineage.

Outbreak of neonatal listeriosis associated with mineral oil

In June, 1989, an outbreak of nosocomial listeriosis occurred in Costa Rica. Listeria monocytogenes was isolated from 9 ill infants 4 to 8 days old who were born after the delivery of an infant with early onset listeriosis. One nosocomial infection was fatal, 2 required mechanical ventilation and 1 resulted in hemiparesis. A higher proportion of cases than other infants born during the outbreak were delivered by cesarean section (55% vs. 24%, P = 0.04). Compared with the mothers of 36 random controls, case mothers were more often primiparous (odds ratio, 6.2, P = 0.03) or received general anesthesia before delivery (odds ratio, 4.4, P = 0.09). All infants were bathed with mineral oil from a multidose container. Culture of the oil by cold enrichment grew L. monocytogenes 4b with the same electrophoretic enzyme type as the outbreak strain. We hypothesize that aspiration of contaminated oil may have resulted in systemic listeriosis. General anesthesia may have increased the risk of aspiration. Lung tissue from the infant who died showed lipid-laden macrophages consistent with oil aspiration and had evidence of L. monocytogenes DNA detected by polymerase chain reaction. This is the first nosocomial outbreak of listeriosis in which a common source suggested epidemiologically was microbiologically confirmed. The high attack rate (greater than 200 times the United States rate of perinatal listeriosis) emphasizes the susceptibility of healthy neonates to L. monocytogenes. The results of our study led to the discontinuation of the use of mineral oil for bathing neonates in Costa Rica.

Molecular surveillance of shiga toxigenic Escherichia coli O157 by PulseNet USA.

PulseNet USA is the national molecular subtyping network system for foodborne disease surveillance. Sixty-four public health and food regulatory laboratories participate in PulseNet USA and routinely perform pulsed-field gel electrophoresis of Shiga toxigenic Escherichia coli isolated from humans, food, water, and the environment on a real-time basis. Clusters of infection are detected in three ways within this system: through rapidly alerting the participants in the electronic communication forum, the PulseNet Web conference; through cluster analysis by the database administrators at the coordinating center at the Centers for Disease Control and Prevention of the patterns uploaded to the central server by the participants; and by matching profiles of strains from nonhuman sources with recent human uploads to the national server. The strengths, limitations, and scope for future improvements of PulseNet are discussed with examples from 2002. In that year, notices of 30 clusters of Shiga toxigenic E. coli O157 infections were posted on the Web conference, 26 of which represented local outbreaks, whereas four were multistate outbreaks. Another 27 clusters were detected by central cluster detection performed at the Centers for Disease Control and Prevention, of which five represented common source outbreaks confirmed after finding an isolate with the outbreak pattern in the implicated food. Ten food isolates submitted without suspicion of an association to human disease matched human isolates in the database, and an epidemiologic link to human cases was established for six of them.