Detection of genes coding for listeriolysin and Listeria monocytogenes antigen A (ImaA) in Listeria spp. by the polymerase chain reaction.

Two pairs of synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) protocol to detect targeted sequences in genes coding for listeriolysin O and Listeria monocytogenes antigen A (ImaA). Strains of Listeria spp. used in this study were isolated from clinical specimens, contaminated foods, and environmental sources. Primers were targeted to internal regions of the genes coding for listeriolysin (hlyA) and Listeria antigen (ImaA) and amplification fragments were detected after the PCR by agarose gel electrophoresis. PCR was performed using nucleic acids extracted from a collection of 74 strains of Listeria spp. including 18 reference strains, 41 L. monocytogenes, nine L. innocua, five L. seeligeri and one L. ivanovii, encompassing representative sources, serovars, and enzyme electrophoretic types. Although the listeriolysin gene was found exclusively in L. monocytogenes, some strains of serovar 4c were negative. Simultaneous presence of both genes was restricted to L. monocytogenes strains of serovars 1/2, 3, and 4. The ImaA gene was identified in five of 10 L. innocua strains and one L. ivanovii isolated from pork. Strains of L. seeligeri, L. welshimeri, and L. grayi were negative for both genes. The detection limits in the PCR were found to be 10 pg of nucleic acids for the hlyA gene and 1 pg for the ImaA gene.

Use of chemotaxonomy as an aid to differentiate among Capnocytophaga species, CDC group DF-3, and aerotolerant strains of Leptotrichia buccalis.

Four strains of fastidious gram-negative rods, thought to be Capnocytophaga species (formerly CDC group DF-1 or Bacteroides ochraceus) or CDC group DF-3 on the basis of conventional phenotypic criteria, were also analyzed for cellular fatty acid (CFA) composition. It was found that the CFA compositions of these strains were qualitatively incorrect for those taxa. Subsequently, it was determined that all four bacteria were in fact aerotolerant strains of Leptotrichia buccalis, based on biochemical reactions, CFA composition, and lactic acid as the major end product of glucose fermentation. It is recommended that, in addition to conventional cultural and biochemical criteria, all strains of Capnocytophaga or CDC group DF-3 should also be tested for metabolic end products of fermentation and CFA composition as essential adjuncts for identification.

Detection of genes for enterotoxins, exfoliative toxins, and toxic shock syndrome toxin 1 in Staphylococcus aureus by the polymerase chain reaction.

Eight pairs of synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) protocol to detect genes for staphylococcal enterotoxins A to E, exfoliative toxins A and B, and toxic shock syndrome toxin 1 in Staphylococcus aureus strains isolated from clinical specimens and contaminated foods. Primers were targeted to internal regions of the toxin genes, and amplification fragments were detected after the PCR by agarose gel electrophoresis. Unequivocal discrimination of toxin genes was obtained by the PCR by using nucleic acids extracted from 88 strains of S. aureus whose toxigenicity was established biologically and immunologically. In immunological assays, two strains of S. aureus produced equivocal results for production of enterotoxin C or toxic shock syndrome toxin 1, giving an overall concordance between phenotypic and genotypic identification of 97.7%. Primer specificity was established in the PCR by using nucleic acids from known toxin-producing bacterial pathogens and from nontoxigenic S. aureus. Strains of Streptococcus spp., including some producers of pyrogenic exotoxin A carrying the speA gene, were negative by the PCR designed to detect staphylococcal toxins. The detection limits were established for all the staphylococcal toxin genes within their respective PCR protocols. The identification of staphylococcal toxin genes in strains of S. aureus by the PCR offers a very specific, sensitive, relatively rapid, and inexpensive alternative to traditional immunological assays which depend on adequate gene expression for reliability and sensitivity.

Rhodococcus infection of the skin with lymphadenitis in a nonimmunocompromised girl.

A 7-year-old girl had a 4 X 3 X 3 cm nodule on the left wrist with axillary lymphadenopathy. Acid-fast bacilli were seen on a smear from a biopsy specimen of this granulomatous skin lesion. A Rhodococcus species grew on culture. Skin infections caused by Rhodococcus may be more common than the few prior case reports suggest.

Cellular fatty acid composition as an adjunct to the identification of asporogenous, aerobic gram-positive rods.

Cellular fatty acid (CFA) compositions of 561 asporogenous, aerobic gram-positive rods were analyzed by gas-liquid chromatography as an adjunct to their identification when grown on blood agar at 35 degrees C. The organisms could be divided into two groups. In the first group (branched-chain type), which included coryneform CDC groups A-3, A-4, and A-5; some strains of B-1 and B-3; "Corynebacterium aquaticum"; Brevibacterium liquefaciens; Rothia dentocariosa; and Listeria spp., the rods had sizable quantities of antiesopentadecanoic (Ca15:0) and anteisoheptadecanoic (Ca17:0) acids. Other species with these types of CFA included B. acetylicum, which contained large amounts of isotridecanoic (Ci13:0) and anteisotridecanoic (Ca13:0) acids. CFAs useful for distinguishing among Jonesia denitrificans, Oerskovia spp., some strains of CDC groups B-1 and B-3, Kurthia spp., and Propionibacterium avidum were hexadecanoic (C 16:0) acid, isopentadecanoic (Ci15:0) acid, and Ca15:0). The second group (straight-chained type), which included Actinomyces pyogenes; Arcanobacterium haemolyticum; C. bovis; C. cystitidis; C. diphtheriae; C. flavescens, "C. gentalium"; C. jeikeium; C. kutscheri; C. matruchotii; C .minutissimum; C. mycetoides; C. pilosum; C. pseudodiphtheriticum; "C. pseudogenitalium"; C. pseudotuberculosis; C. renale; CDC groups 1, 2, ANF-1, D-2, E, F-1, F-2, G-1, G-2, and I-2; C. striatum; "C. tuberculostearicum"; C. ulcerans; C. vitarumen; C. xerosis; and Erysipelothrix rhusiopathiae, was typified by significant quantities of hexadecanoic (C16:0) and oleic acids (C18:cis9), with differences in the amounts of linoleic acid (C18:2), stearic acid (C18:0), an unnamed peak (equivalent chain length, 14.966), and small quantities of other known saturated and unsaturated fatty acids. CFA composition of these organisms was sufficiently discriminatory to assist in classification but could not be used as the sole means of identification.

Alteration of phage- and biotypes of Listeria strains.

Ethyl-methyl-sulphonate mutants of Listeria monocytogenes might be different from the parent strain in phage type and in splitting of lactose, maltose, melezitose, sucrose and trehalose. Differences were observed in repeated control studies in phage type and carbohydrate-decomposition of 550 Listeria strains isolated from a variety of sources (patients, healthy and dead animals). It has been concluded that certain carbohydrate tests are unsuitable for distinguishing biotypes of Listeria. An improvement of the evaluation of phage typing results is recommended.

Listeria monocytogenes infections in Canada.

Since its first isolation by Murray in 1926 Listeria monocytogenes has become recognized as a significant pathogen occurring worldwide and involving a wide range of wild and domestic animals including man. The first confirmed human listeriosis case in Canada was published by Stoot in 1951. A later survey based on records maintained at the Laboratory Centre for Disease Control, Ottawa showed 101 cases detected over a 21 year period in nine provinces. The overall mortality was 30%. The most frequently isolated serotype was 4b followed by 1 and 1b. Prior to the Nova Scotia epidemic (41 cases) of 1981, fewer than 15 cases per annum had been diagnosed based on hospital discharge records. The Nova Scotia epidemic was unique in that the source and mode of transmission of the organism were determined. Sixty-three strains isolated from this outbreak were typed, and with the exception of one 1a strain, were identified as 4b. These were subsequently classified mainly as phage type 00 042 0000 and 00 002 0000. Listeriosis appears to be a common infection in the animal population in Canada primarily in cattle, sheep, chinchillas, chickens and goats. Outbreaks have been described in rabbits, goats, and chinchillas. Chinchilla farms were affected in one outbreak (serotype 1) in Nova Scotia which was attributed to feeding a new batch of meal containing beet pulp. Many aspects of the epidemiology of listeriosis are obscure. A cycle involving contaminated soil and consumption of raw vegetables has been confirmed as the cause of the Nova Scotia epidemic and could explain a proportion of the sporadic cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Surveillance of Listeria monocytogenes in human and environmental specimens in Nova Scotia, 1974 to 1981.

We examined specimens for L. monocytogenes using the "cold enrichment" technique of Gray et al. (J. Bacteriol., 55: 471, 1948) and a nalidixic agar plate (Ann. Inst. Pasteur 111: 90, 1966). Between 1974 and May 1981, we isolated L. monocytogenes from four of 5,255 specimens (rectal, vaginal and placental swabs; blood; spinal fluid; semen; necropsy material) which came from eight human populations (neonates, children, adult men, and pregnant and nonpregnant women) and from 161 animals. Three of the isolated strains were type 1, and they came from a newborn born at 32 weeks' gestation, that child's mother, and another woman who had recently delivered. The fourth (type 4b) came from a newborn twin born at 36 1/2 weeks' gestation. In June through October 1981, in 529 specimens, we isolated L. monocytogenes type 1/2 from two of four larvae tested (four earwigs and five slugs were all negative) and from three of 27 samples of fresh chicken liver (however, 18 samples of coleslaw were negative). At the same time, we isolated L. monocytogenes (not yet typed) from a rectal swab from one of 112 dogs examined. Rectal swabs from 107 cats were negative, as were vaginal swabs from 144 women and urine samples from 108 newborns.