Sepsis due to coryneform group A-4 in an immunocompromised host.

Corynebacteria are more commonly being recognized as significant human pathogens. We describe a case of Coryneform group A-4 sepsis secondary to infection of a Hickman catheter in an immunocompromised man; the organism was identified by biochemical analysis conducted at the Louisiana State Reference Laboratory.

Reversed-phase liquid chromatographic determination of vitamin D in milk.

Vitamin D in milk is determined by a slight modification of the method of Sliva et al. [J. AOAC Int. (1992) 75, 566-571] for infant formula and enteral nutritional products. The sample is saponified for 30 min at 60 degrees C and extracted overnight into 60 mL of hexane. The hexane layer is washed, neutralized, and taken to dryness with a rotary evaporator. The sample is reconstituted in hexane and applied to 500 mg of Florisil in a solid-phase extraction column. Vitamin D is eluted with isopropyl alcohol. The eluate is evaporated to dryness under N2, and the sample is reconstituted in 1.0 mL of acetonitrile. The extract is analyzed on a C18 liquid chromatographic column (250 x 4.6 mm, 5 microns particle size) with UV detection at 265 nm. Milk samples of various fat content (i.e., skim, low fat, and whole milk) were analyzed. Spiked recoveries gave means of 81-96%; recoveries were inversely related to fat content. Assay precision ranged from 3.2 to 8.6%. The method can measure vitamins D2 and D3 individually, and no difference in the recoveries of the 2 vitamins was observed. Thus, vitamin D2 can be used as an internal standard for quantitating vitamin D3, and vice versa. The method is satisfactory for use in screening of milk for vitamin D content.

New developments in mycobacteria identification: public health laboratory modernization.

Clinical laboratory mycobacteriology has traditionally involved long delays for results, primarily due to the slow growth rate of most members of the genus Mycobacterium. Several new methodologies are now available that enable dramatic reductions in turn-around times. These methodologies include the Bactec TB System for isolation and drug susceptibility testing of Mycobacterium tuberculosis, the use of DNA probes, and high performance liquid chromatography (HPLC) for the identification of mycobacterial isolates. The spread of multidrug-resistant tuberculosis has made it imperative that laboratories take advantage of these new methodologies whenever possible. The Mycobacteriology Unit of the Louisiana Office of Public Health's Division of Laboratories has pioneered the use of these methodologies in a public health laboratory setting. Great reductions in turn-around times have been achieved and further reductions are expected as the methods are refined and adapted to the needs of a high-volume public health laboratory.

Vibrio gastroenteritis in Louisiana: a prospective study among attendees of a scientific congress in New Orleans.

The incidence of diarrhea associated with infection by Vibrio species was investigated among attendees at the 1986 Interscience Conference on Antimicrobial Agents and Chemotherapy held in New Orleans. Twelve percent of respondents reported diarrhea; the risk of diarrhea was significantly higher in those who ate raw or cooked oysters (relative risk = 1.5, P = .005). At least one Vibrio species was recovered from 51 (11%) of 479 persons submitting stool specimens; however, only 15 (29%) of those with a positive stool culture also reported diarrhea. Of the five Vibrio species identified, V. parahaemolyticus was most common and was most strongly associated with diarrhea. V. cholerae serogroup O1 was not isolated despite the occurrence of a cholera outbreak during the same time period in Louisiana. Cultures of raw and cooked seafood served in local restaurants yielded five different Vibrio species. Although asymptomatic passage of Vibrio organisms was common among persons eating seafood, the risk of Vibrio gastroenteritis was low.

Cholera in Louisiana. Widening spectrum of seafood vehicles.

The largest cholera outbreak in the United States in over a century occurred in Louisiana from August through October 1986. Eighteen persons in 12 family clusters had stool culture or serologic evidence of infection with toxigenic Vibrio cholerae 0-group 1. Thirteen of these persons had severe diarrhea, and 4 required intensive care unit treatment. Although all 18 survived, 1 96-year-old woman with suspected cholera died shortly after hospital admission. A case-control study showed that case-patients were more likely than neighborhood control subjects to have eaten cooked crabs or cooked or raw shrimp during the week before illness. Case-patients who ate crabs were more likely than control subjects who ate crabs to have undercooked and mishandled the crabs after cooking. A third vehicle from the Gulf waters, raw oysters, caused V cholerae 01 infection in two persons residing in Florida and Georgia. All three seafood vehicles came from multiple sources. Stool isolates from the Louisiana case-patients were genetically identical to other North American strains isolated since 1973, but differ from African and Asian isolates. While crabs are the most important vehicle for V cholerae 01 infection in the United States, shrimp and oysters from the Gulf coast can also be vehicles of transmission. A persisting reservoir of V cholerae 01 along the Gulf coast may continue to cause sporadic cases and outbreaks of cholera in Gulf states and in states importing Gulf seafood.

Molecular epidemiology of non-O1 Vibrio cholerae and Vibrio mimicus in the U.S. Gulf Coast region.

Ten toxigenic Vibrio cholerae non-O1 and V. mimicus strains isolated from clinical and environmental sources in the U.S. Gulf Coast region were examined for genetic relatedness. Restriction digest patterns of chromosomal DNA and Southern blot analysis with a cholera toxin gene probe revealed that the strains exhibited greater genetic divergence than the highly conserved V. cholerae O1 strains isolated from clinical and sewage samples in this region.

Broth medium for enrichment of Vibrio fluvialis from the environment.

A medium was designed for the enrichment and enumeration of Vibrio fluvialis from environmental samples. The medium contains 1% peptone plus 4% sodium chloride and 5 micrograms of novobiocin per ml, pH 8.5. This V. fluvialis enrichment medium (FEM) was tested, in comparison with alkaline peptone (AP), in field samplings. A total of 177 samples (estuarine waters and sediment, sewage, and crabs) collected over a 14-month period were examined with FEM and with AP broth. Results showed that FEM was more effective than AP in detecting V. fluvialis, particularly from water and sewage samples with low salinities (less than 6%). The best recovery of V. fluvialis occurred when both enrichment media were used simultaneously.

Isolation of nontoxigenic Vibrio cholerae O1 from a human wound infection.

Vibrio cholerae serotype O1 organisms that do not produce cholera toxin and, in fact, lack the genetic material encoding the enterotoxin have recently been detected in coastal regions of the United States. Although these organisms have been assumed to be nonpathogenic, they have been considered a potential reservoir of toxigenic V. cholerae. In 1979, nontoxigenic V. cholerae O1 was isolated from a leg wound of an accident victim residing in New Orleans. The only known risk factors of the patient, besides his debilitated condition, were alcoholism and the consumption of raw oysters before recognition of his wound infection. Coincident with the identification of the isolate from the leg wound, an identical nontoxigenic V. cholerae O1 isolate was cultured from the sewage system serving the residence of this patient. Nontoxigenic V. cholerae O1 seems to be capable of multiplying in human tissue and may produce extraintestinal infection. This indigenous inhabitant of temperate coastal regions may not be avirulent and may be of public health significance.

Molecular epidemiology of Vibrio cholerae in the U.S. Gulf Coast.

Enterotoxigenic strains of Vibrio cholerae O-1, biotype El Tor, isolated from a case of cholera in Texas in 1973, an outbreak of cholera in Louisiana in 1978, and Louisiana sewage samples in 1980 and 1981 were analyzed for their genetic similarities. Chromosomal DNA was isolated from each strain, digested with restriction endonuclease, and analyzed by the Southern blot technique. A radioactive probe consisting of Escherichia coli heat-labile enterotoxin DNA detected cholera toxin gene sequences in these strains and demonstrated that the toxin gene sequence, if not the entire chromosomal DNA, is identical in these strains and distinctly different from other strains of V. cholerae isolated throughout the world. In addition, two strains of enterotoxigenic V. cholerae non-O-1 isolated from clinical cases, were analyzed and found to possess cholera toxin genes which differed in the DNA sequence from the V. cholerae O-1 strains. We concluded that a single strain of enterotoxigenic V. cholerae O-1 is resident in the U.S. Gulf Coast and that a second reservoir of cholera toxin genes exists in V. cholerae non-O-1 strains in Louisiana.

Vibrios in the Louisiana gulf coast environment.

A polyphasic approach, using bacteriological, immunological, and molecular biological techniques was used to elucidate the distribution of pathogenicVibrio species in the Louisiana coastal environment. A variety ofVibrio species pathogenic for man, includingV. cholerae, V. parahaemolyticus, V. fluvialis, andV. vulnificus, were found to be ubiquitous in Louisiana.Vibrio species monitored were shown to fluctuate in response to environmental factors of temperature, salinity, and nutrient level, and to vary independently of fecal coliform counts. A comprehensive serological screening system, based on species specific H antigens, was developed to identify pathogenicVibrio sp. 1 step after primary isolation.Vibrio sp. were correctly identified with accuracies ranging from 93-100%, depending on the specific H antiserum. Over 2,500V. cholerae isolates were rapidly screened for production of cholera toxin by DNA hybridization of specific toxin gene probes to colonies inoculated on nitrocellulose filter paper. The toxin gene probes, together with O antigen analysis, revealed that enterotoxigenicV. cholerae 01 serovars were recovered only from sewage stations or human disease, whereas enterotoxigenicV. cholerae non 01 serovars were recovered from environmental samples in addition to clinical and sewage samples. The results of this study indicate that techniques of immunology and molecular biology are very valuable supplements to conventional bacteriological techniques in studying the epidemiology and ecology of pathogenicVibrio sp.

Characterization of biochemically atypical Vibrio cholerae strains and designation of a new pathogenic species, Vibrio mimicus.

Biochemically atypical strains classified as Vibrio cholerae were characterized by biochemical reactions, serology, antibiotic susceptibility testing, and deoxyribonucleic acid relatedness. Strains with the following atypical reactions were shown to be V. cholerae: mannose negative, mannitol negative, lysine decarboxylase negative, no growth in the presence of 5% NaCl, salicin and cellobiose positive. Sucrose-negative strains were shown to constitute a new species, Vibrio mimicus, whose type strain is 1721-77 (ATCC 33653). In addition to its negative sucrose reaction, V mimicus was differentiated from V. cholerae by its negative Voges-Proskauer, corn oil, and Jordan tartrate reactions and by its sensitivity to polymyxin. V. mimicus was isolated from shellfish and water, as well as from human diarrheal stools and ear infections. Most strains were typable with antisera against V. cholerae. Strains from three serogroups produced either a heat-labile or a heat-stable enterotoxin.

Use of Moore swabs for isolating Vibrio cholerae from sewage.

The Moore swab method was shown to be a practical and sensitive technique for the isolation of Vibrio cholerae from sewage. In each of three instances in which cholera patients lived in homes connected to municipal sewers, V. cholerae was isolated from the community sewage plant intake at the time of the patients illness. Sewer systems became negative within 1 day after patients were treated with tetracycline. Sewer surveillance using the Moore swab also found evidence of infections occurring in areas where surveillance of diarrheal illness failed to detect cholera. Culturing community sewage by the Moore swab method proved to be an economical and effective way of determining areas where V. cholerae infections were occurring.

A unique environmental occurrence of hydrogen sulfide positive Escherichia coli.

For the first time in nearly 4 decades of surveillance, H2S positive Escherichia coli have been isolated from Calcasieu Lake and River. These results are reported because of recent clinical interest in these organisms.

Some biological and physical properties of molluscum contagiosum virus propagated in cell culture.

Molluscum contagiosum virus propagated in FL cells of human amnion origin has a one-step growth cycle time of 12 to 14 h. The appearance and exponential increase of intracellular virus preceded the release of extracellular virus by approximately 2 h. Demonstration of comparable titers of extracellular and intracellular virus at the end of the replication cycle indicated that a substantial amount of virus remained associated with cells exhibiting cytopathogenic changes. Mean buoyant density values of virus in sucrose ranged from 1.275 to 1.278 g/cm3, but in CsCl the virus banded at densities at 1.325 to 1.340 and 1.261 to 1.281 g/cm3. Although virus infectivity was not affected by high concentrations of CsCl, it was found by polyacrylamide gel electrophoresis that the salt removed several nonglycosylated polypeptides with estimated molecular weights of 15,000 to 60,000. This suggested that the high-density band (1.325 to 1.340) may reflect the loss of these structural components. The half-life of virus infectivity was approximately 26.5 h at 26 degrees C and 11.2 h at 37 degrees C. Although the virus was rapidly inactivated at 50 degrees C, it could be stabilized at this temperature by the presence of 1.0 M MgCl2. Virus did not agglutinate newborn chick, adult chicken, or type "0" human erythrocytes. Virus infectivity was found to be sensitive to acid pH but resistant to treatment with diethyl ether or chloroform. The replication of molluscum virus in FL cells was not inhibited by 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, or cytosine arabinonucleoside in noncytotoxic concentrations of 200 to 400 mug/ml, but greater than 99% reduction in the yield of herpes simplex virus or vaccinia virus in FL cells was obtained with 200 mug of these compounds per ml. Guanidinium chloride in concentrations of 100 to 200 mug/ml reduced molluscum virus yields by more than 99.9%.