Diagnostic value of plasmid analyses and assays for virulence in Yersinia enterocolitica.

The possession of a 42- to 48-megadalton plasmid alone does not appear to be predictive of virulence in Yersinia species. Twelve of 100 Yersinia enterocolitica strains contained a 42 to 48-megadalton plasmid, and 4 of 30 Y. enterocolitica-like strains contained a 42- to 48-megadalton plasmid. Seven strains of Y. enterocolitica contained the 42- to 48-megadalton plasmid plus an 82-megadalton plasmid, and these were the only study strains lethal for mice. Based on restriction endonuclease digestion, the 42- to 48-megadalton plasmid DNA from these seven strains were similar and were not similar to the 42- to 48-megadalton plasmids present in the other nine strains. The ability to invade guinea pig eye tissues, calcium dependency, autoagglutination, and colonial morphology at 37 degrees C were also associated with plasmid DNA, but the relationships were either variable or not reciprocal. Neither tissue culture invasiveness nor heat-stable toxin production was associated with plasmid DNA. It was concluded that biochemical speciation and a total plasmid profile in combination with enzyme digests are predictive of virulence in Y. enterocolitica as it is measured by mouse lethality.

Evaluation of the chick embryo for the determination of relative virulence of Neisseria meningitidis.

The chick embryo model was evaluated as a method to compare virulence between selected strains of Neisseria meningitidis. Inoculation of 13-day-chick embryos via the egg yolk distinguished strains having an LD50 of 10(3) colony forming units (CFU) or greater (low virulence) from those having an LD50 of approximately 10(1) or less (high virulence). A strain of serogroup B and a spontaneous nonpiliated strain of group C were found to be of relatively high virulence while a strain of N. lactamica, a serogroup A carrier strain, and certain nongroupable strains were found to be of low virulence. Strains having an LD50 of 10(2) were not differentiated from either of these. Alternatively, inoculation of the chorioallantoic membrane (CAM) of 9-day-old chick embryos statistically differentiated most strains of N. meningitidis although inoculation via this route was less sensitive.

Changes in the virulence of Mycobacterium avium after passage through embryonated hens' eggs.

Eight-day-old embryonated hen's eggs were used as a model to study Mycobacterium avium virulence. Strains isolated from human patients caused 20-90% mortality when eggs were infected by injection of bacterial suspensions into the amniotic sac. Virulence of examined strains subsequently decreased with passage through eggs to between 0 and 40% mortality in four passages. Virulence of the egg-attenuated strains could be restored by passage through human peripheral blood mononuclear cells. The site of infection in the egg was usually the mesodermal layer of the chorioallantoic membrane. A few small granulomas containing acid-fast bacteria were seen in the liver, but not in other organs. Death of chicken embryos may have resulted from destruction of the mesodermal layer of the chorioallantoic membrane with consequent respiratory failure. PBMCs infected with less virulent egg-passaged strains of M. avium produced higher levels of tumor necrosis factor-alpha than did peripheral blood mononuclear cells infected with more virulent nonpassaged strains.

Mycobacterium xenopi multiplies within human macrophages and enhances HIV replication in vitro.

Mycobacterium xenopi can cause opportunistic infections, particularly in persons infected with human immunodeficiency virus type 1 (HIV-1). The primary focus of this effort was to determine if M. xenopi isolates could survive and grow in human peripheral blood macrophage (MPhi), and if these isolates could promote the replication of HIV-1 in vitro. M. xenopi bacilli survived and replicated 10-fold within 48 h in human MPhi while avirulent Mycobacterium smegmatis, did not grow within the MPhi. M. xenopi bacilli when cultured with peripheral blood mononuclear cells enhanced HIV-1 replication 30- and 50-fold with the macrophage-tropic HIV-1(Ba-L) and 50- and 75-fold with T-cell-tropic strain HIV-1(LAI) by 6 days post-infection when compared to M. smegmatis. The enhanced HIV replication was associated with increased production of TNF-alpha. Partial inhibition of HIV-1 induction was observed using a neutralizing anti-TNF-alpha monoclonal antibody, pentoxifylline, and matrix metalloproteinase (MMP) inhibitor I. Similar mechanisms of pathogenesis among mycobacterial species may help elucidate better treatment approaches in HIV co-infected persons.

In vitro antimicrobial susceptibility, plasmid analysis, and serotyping of epidemic-associated Campylobacter jejuni.

Campylobacter jejuni strains from 11 outbreaks were characterized by antimicrobial susceptibility, plasmid profile, and serotyping by the methods of Lior et al. and Penner and Hennessy. All 31 strains were susceptible to erythromycin, clindamycin, chloramphenicol, kanamycin, tobramycin, streptomycin, and gentamicin. A total of 21 strains from nine outbreaks were resistant to one or more of the following antimicrobial agents: tetracycline, metronidazole, ampicillin, or carbenicillin. Of the 31 strains, 19 possessed plasmid DNA; 4 of the strains containing plasmids were sensitive to all antimicrobial agents tested. All of the strains that were resistant to tetracycline contained a 38-megadalton plasmid, and these plasmids shared common nucleic acid sequences. No other antimicrobial resistance was associated with the presence of plasmid DNA. Eight outbreaks appeared to have been caused by a single serotype, whereas in three outbreaks multiple serotypes were found. In two of the three outbreaks with multiple serotypes, plasmid profiles were also indicative of multiple strains of C. jejuni. Antimicrobial susceptibility and plasmid profile are potentially useful epidemiological markers for C. jejuni and may be used to supplement serotyping.

Laboratory observations on Plesiomonas shigelloides strains isolated from children with diarrhea in Peru.

Eleven strains of Plesiomonas shigelloides isolated from 10 Peruvian children with diarrhea were examined. All the strains were resistant to two or more antibiotics, most commonly ampicillin, gentamicin, erythromycin, kanamycin, and streptomycin. The strains were all negative in the Sereny and cell culture assays used to test for enteroinvasiveness. One strain showed cytotoxic activity on Vero cells. The strains showed no antigenic relationship with Shigella organisms. Both bioassays and enzyme-linked immunosorbent assays used for detection of Escherichia coli enterotoxins were negative. Nucleic acid probes for such toxins likewise gave negative results. The strains all possessed a large (approximately 200-megadalton) plasmid in addition to one or more other plasmids. Several different plasmid profiles were observed among these 11 P. shigelloides strains, indicating that the isolates were not acquired from a common source or from a single bacterial clone.

Isoprenoid quinones of "Afipia" spp.

The isoprenoid quinone contents of seven strains of "Afipia felis," the type strains of "A. clevelandensis" and "A. broomeae," and reference strains of three unnamed "Afipia" genospecies were determined by reverse-phase high-performance liquid chromatography. The quinone profiles of all "Afipia" strains were essentially identical, with ubiquinone 10 as the major component. The identity of ubiquinone 10 was confirmed by mass spectrometry.

Mycobacterium avium bacilli grow saprozoically in coculture with Acanthamoeba polyphaga and survive within cyst walls.

Protozoans are gaining recognition as environmental hosts for a variety of waterborne pathogens. We compared the growth of Mycobacterium avium, a human pathogen associated with domestic water supplies, in coculture with the free-living amoeba Acanthamoeba polyphaga with the growth of M. avium when it was separated from amoebae by a 0.1-micron-pore-size polycarbonate membrane (in a parachamber). Although viable mycobacteria were observed within amoebal vacuoles, there was no significant difference between bacterial growth in coculture and bacterial growth in the parachamber. This suggests that M. avium is able to grow saprozoically on products secreted by the amoebae. In contrast, Legionella pneumophila, a well-studied intracellular parasite of amoebae, multiplied only in coculture. A comparison of amoebae infected with L. pneumophila and amoebae infected with M. avium by electron microscopy demonstrated that there were striking differences in the locations of the bacteria within amoebal cysts. While L. pneumophila resided within the cysts, M. avium was found within the outer walls of the double-walled cysts of A. polyphaga. These locations may provide a reservoir for the bacteria when environmental conditions become unfavorable.

Salmonellosis at rural and urban clinics in Bangladesh: epidemiologic and clinical characteristics.

The authors studied the frequency of diarrheal illness associated with non-typhi Salmonella at two clinics in Bangladesh for the years 1977-1979. Non-typhi salmonellae were isolated from 0.29% of fecal specimens or rectal swabs in an urban area and 0.26% of similar specimens in a rural area; the frequency of isolations peaked in the summer months. Isolations of Shigella and Vibrio cholerae were much more common than Salmonella. Only two of 50 Salmonella isolates were resistant to more than one antibiotic. None of 13 isolates tested produced an enterotoxin. S. java and S. virchow accounted for 64% of all the isolates. Patients with diarrheal illness associated with isolation of Salmonella frequency had vomiting (88%), watery diarrhea (78%), abdominal pain (61%), and fever (39%), but the clinical features of the illnesses and the socioeconomic backgrounds of the patients could not be distinguished from those of matched controls who were attending the same clinic. The infrequency of Salmonella infection in an area where several other bacterial and viral enteric diseases are hyperendemic requires further investigation.

Genetic transfer of antimicrobial resistance and enterotoxigenicity among Escherichia coli strains.

To understand the role of enterotoxin (Ent) plasmids in epidemics of enterotoxigenic (ET) Escherichia coli diarrhea in the United States, we studied the genetics of Ent plasmids in relation to E. coli serotypes and R plasmids. Twenty-nine ET E. coli strains, including all epidemic isolates available at the Centers for Disease Control, Atlanta, Ga. (CDC), were assessed for the ability to transfer antimicrobial resistances (if present) by conjugation, to mobilize a nonconjugative R plasmid, and to cotransfer enterotoxigenicity with R determinants. Of the 12 ET E. coli strains isolated in the United States, 5 were able to transfer R plasmids; one strain cotransferred detectable enterotoxigenicity. Another four U.S. isolates were able to mobilize plasmid DNA, but no toxin production was detected in transconjugants. Of 17 resistant ET E. coli from South Asia, 13 were able to transfer R plasmids; 5 of those 13 cotransferred detectable Ent plasmids. In all, 22 ET E. coli strains (76%) were able to initiate conjugation and genetic transfers. Six of these strains (20%) were able to cotransfer enterotoxigenicity with a conjugative R plasmid at a detectable frequency. One of the six strains transferred R and Ent determinants on a single plasmid. These data are addressed in relation to the observed immobility of Ent and R during outbreaks of ET E. coli, the efficacy of prophylactic tetracycline, and the worldwide occurrence of a limited number of ET E. coli serotypes.

Persistence of plasmids, cholera toxin genes, and prophage DNA in classical Vibrio cholerae O1.

Plasmid profiles, the location of cholera toxin subunit A genes, and the presence of the defective VcA1 prophage genome in classical Vibrio cholerae isolated from patients in Bangladesh in 1982 were compared with those in older classical strains isolated during the sixth pandemic and with those in selected eltor and nontoxigenic O1 isolates. Classical strains typically had two plasmids (21 and 3 megadaltons), eltor strains typically had no plasmids, and nontoxigenic O1 strains had zero to three plasmids. The old and new isolates of classical V. cholerae had two HindIII chromosomal digest fragments containing cholera toxin subunit A genes, whereas the eltor strains from Eastern countries had one fragment. The eltor strains from areas surrounding the Gulf of Mexico also had two subunit A gene fragments, which were smaller and easily distinguished from the classical pattern. All classical strains had 8 to 10 HindIII fragments containing the defective VcA1 prophage genome; none of the Eastern eltor strains had these genes, and the Gulf Coast eltor strains contained a different array of weakly hybridizing genes. These data suggest that the recent isolates of classical cholera in Bangladesh are closely related to the bacterial strain(s) which caused classical cholera during the sixth pandemic. These data do not support hypotheses that either the eltor or the nontoxigenic O1 strains are precursors of the new classical strains.

An outbreak of salmonellosis associated with a fatality in a healthy child: a large dose and severe illness.

In June 1982, an outbreak of Salmonella gastroenteritis occurred on a farm in Wyoming. All eight affected persons became severely ill 8-18 hours after they had eaten homemade ice cream. A previously healthy 13-year-old boy died 37 hours after exposure; his mother and four younger siblings were transferred to intensive care units in hospitals in adjoining states, and the remaining two adult males were hospitalized locally. Salmonella typhimurium was isolated from all eight ill persons, from the remaining ice cream, and from the family's hens whose eggs were used in the preparation of the ice cream. All Salmonella contained identical plasmids (60-, 5.6-, and 3.3-megadalton); the ice cream contained 10(6) salmonellae/g and, according to food histories, individuals consumed an estimated dose of between 10(8) and 10(9) organisms. The fatal illness occurred in the boy who had eaten the largest amount of ice cream (10(9) organisms). This report demonstrates that Salmonella can cause fatal illness in previously healthy individuals and that the incubation period and the severity of the illness may be related to the dose.

A tissue culture bilayer model to study the passage of Neisseria meningitidis.

A tissue culture bilayer system has been developed as a model to study the mechanisms of attachment and invasion involved in the pathogenesis of Neisseria meningitidis. The model incorporates epithelial and endothelial cell layers separated by a microporous membrane and makes it possible to observe and quantify the passage of bacteria through the multiple layers and to study the mechanisms by which they make this passage. This model is adaptable to a wide variety of microbial pathogens and can be modified by substituting any physiologically relevant eucaryotic cells for the component layers. The system's makeup of cells of human origin and its reproducibility give it advantages over animal and primary organ culture models, while the added complexity of multiple layers allowing cell-to-cell communication makes it a more realistic human tissue model than standard cell monolayers.

Plasmid characterization of Salmonella typhimurium transmitted from animals to humans.

The transmission of pathogenic bacteria from animals to humans is widely studied because of its public health importance. In this study, we show the transmission of Salmonella typhimurium from cattle which had received no growth-promoting antibiotics to humans who had direct contact with the ill animals. On one cattle farm, the veterinarian attending the sick animals became ill, and two other individuals living on the farm later developed salmonellosis. The strains isolated from both humans and animals at one farm were identical as to antibiotic susceptibility and phage type, and they were specifically traced by the presence of a common 24-megadalton plasmid. Restriction enzyme digests of this plasmid from both human and animal strains were identical. At another farm, tetracycline-resistant S. typhimurium strains possessing a different profile (eight plasmids) were isolated from both animals and humans. The tetracycline-resistant clone was also isolated from animals at a third farm, but with animals and humans having no known contact with those of the other two farms.