Isolation of Shigellae: V. Comparison of Enrichment Broths with Stools

Many enteric media are more efficient for the detection of salmonellae than of shigellae. Comparisons of three enrichment broths and three plating media were made during analysis of 1,405 stool specimens to choose a combination of media which would enhance detection of shigellae as well. Gram-Negative (GN), Selenite, and Silliker's Broths were streaked to E M B, Salmonella-Shigella (SS), and xylose lysine deoxycholate (XLD) Agars. The enrichment broths produced a twofold increase in isolations of both salmonellae and shigellae over direct streaking. All three broths performed equally well for Salmonella detection, but GN and Silliker's produced twice as many Shigella isolates as did Selenite. Comparison of the plating media showed that XLD was markedly more efficient than either E M B or SS Agar for the recovery of both genera. SS Agar was superior to E M B for isolation of salmonellae after enrichment, whereas E M B was better for isolation of shigellae by direct streaking. Both E M B and SS were more effective when used after GN and Silliker's than after Selenite. GN Broth and XLD Agar were the most efficient combination of media. During these analyses, 158 salmonellae and 49 shigellae isolates were obtained.     

Experimental salmonellosis conjunctivitis and keratitis in guinea pigs]

The authors present the results of morphological study of experimentally-induced conjunctivitis and keratitis induced in guinea pigs by salmonellae. It was revealed that injuries of the conjunctiva and cornea in salmonellae infection were different by development and morphological manifestations from keratoconjunctivitis caused by shigellae. This difference was due to specific peculiarities of the pathogenic properties of salmonellae and shigellae clearly expressed on the given experimental model.     

Isolation of Shigellae: VI. Performance of Media with Stool Specimens

The efficiencies of three enrichment broths and four plating media for isolation of enteric pathogens were compared from 1,117 stool specimens. Direct streaking proved to be inferior to enrichment, detecting only 50% of the salmonellae and 61% of the shigellae. By contrast, Selenite Broth (SF) found 90% of the total salmonellae isolates and 82% of the shigellae isolates. Gram-Negative Broth (GN) found 82% and 85%, respectively, but Tetrathionate found only 60% and 39%. Thus, SF and GN were comparable for both salmonellae and shigellae and significantly better than Tetrathionate Broth for both. The plating media compared were MacConkey (MAC), deoxycholate citrate (DC), xylose lysine deoxycholate (XLD), and xylose lysine Brilliant Green (XLBG) Agars. Of the total salmonellae isolated, XLD produced 94%; XLBG, 71%; MAC, 55%; and DC, only 35%. Of shigellae, XLD found 89%; MAC, 75%; XLBG, 63%; and DC, but 27%. The efficacy of XLD is observed to be almost threefold that of DC. The most successful combination of media for the detection of fecal pathogens was GN or SF enrichment broths streaked to XLD plates. These analyses resulted in the isolation of 118 strains of salmonellae and 33 of shigellae.     

Isolation of Shigellae. VII. Comparison of Gram-Negative Broth with Rappaport''s Enrichment Broth

Efficacies of Gram Negative Broth (GN) and Rappaport's Enrichment Broth (RE) were compared for detection of enteric pathogens from clinical specimens. Whereas direct streaking on four plating media found 57% of the salmonellae, GN found 80% and RE found 92% of the 157 isolates. By contrast, direct streaking found 87% of the shigellae whereas GN detected 93%, but RE found only 20%. RE produced 35% more Salmonella-positive plates than GN did, which resulted in a 15% increase in isolates. Statistically, RE proved to be significantly better than GN. The unsuitability of RE for shigellae, however, dictates that, if only one enrichment broth is to be used, GN must be that choice, but that maximal isolations of all enteric pathogens should result from the use of both RE and GN. Of the four plating media, Xylose Lysine Deoxycholate Agar detected 90% of the salmonellae and 85% of the shigellae. Salmonella-Shigella Agar detected 72 and 43%, respectively, and Levine Eosin Methylene Blue Agar found 45 and 55%. Bismuth Sulfite Agar detected only 22% of the salmonellae and found no shigellae. The performance of all of the plating media was enhanced by enrichment, but RE was especially effective for salmonellae when compared to GN.     

Isolation of Shigellae. VII. Comparison of Gram-Negative Broth with Rappaport's Enrichment Broth

Efficacies of Gram Negative Broth (GN) and Rappaport''s Enrichment Broth (RE) were compared for detection of enteric pathogens from clinical specimens. Whereas direct streaking on four plating media found 57% of the salmonellae, GN found 80% and RE found 92% of the 157 isolates. By contrast, direct streaking found 87% of the shigellae whereas GN detected 93%, but RE found only 20%. RE produced 35% more Salmonella-positive plates than GN did, which resulted in a 15% increase in isolates. Statistically, RE proved to be significantly better than GN. The unsuitability of RE for shigellae, however, dictates that, if only one enrichment broth is to be used, GN must be that choice, but that maximal isolations of all enteric pathogens should result from the use of both RE and GN. Of the four plating media, Xylose Lysine Deoxycholate Agar detected 90% of the salmonellae and 85% of the shigellae. Salmonella-Shigella Agar detected 72 and 43%, respectively, and Levine Eosin Methylene Blue Agar found 45 and 55%. Bismuth Sulfite Agar detected only 22% of the salmonellae and found no shigellae. The performance of all of the plating media was enhanced by enrichment, but RE was especially effective for salmonellae when compared to GN.     

Effect of Temperature of Incubation on Performance of Media in the Detection of Enteric Pathogens

The effect of incubation temperatures on the efficacies of both plating media and transport or enrichment broths was determined by the analysis of 391 diarrheal stools for salmonellae and shigellae. Each analysis resulted in 90 observations. Stool specimens were homogenized in saline and used to inoculate eosin methylene blue (EMB), Salmonella-Shigella (SS), and xylose lysine deoxycholate (XLD) agar plates, Amies and Cary-Blair (CB) transport media, and gram-negative (GN) enrichment broth. All media were incubated at 25, 30, and 35 C for 24 and 48 h. In order of efficacy, GN and saline were significantly better than Amies and CB, which were still better than direct streaking for both salmonellae and shigellae. Forty-eight hours was a significant improvement over 24 h only at 25 C on direct streaking for both pathogens. Salmonella detection was also improved at 30 over 25 C on direct streaking. In direct plating, XLD was better than both SS and EMB for both pathogens. After broths, for salmonellae, XLD > SS > EMB, and for shigellae, XLD > EMB > SS, with all differences significant. SS agar was significantly improved for detection of shigellae with 48-h broth inocula versus 24-h broth inocula. The differences thus observed at the various temperatures tested proved to be less important than the media used. The efficient media, GN broth, saline-stool, and XLD were shown to be affected very little by either temperature or time variance of the magnitude tested.     

Mechanism of antagonistic activity of lactobacilli

Different species of lactobacilli, when grown together with salmonellae and shigellae, have been found to inhibit their growth and proliferation. The supernatants of the culture fluids of 8 species of lactobacilli have been shown to contain natural metabolites, other than lactic acid, with a wide spectrum of antibacterial activity with regard to different opportunistic aerobic and anaerobic microorganisms. The relationship between the antagonistic activity and the presence of plasmid DNA in a number of the most active antagonistic lactobacilli is discussed.     

Monitoring of etiological agents of acute enteric infections in the Moscow region

The fecal microflora of patients with acute enteric infections (AEI) has been examined on the territory of the Moscow region. The pathogens of high, moderate and low priority levels have been detected. As revealed in this study, shigellae, salmonellae, enteropathogenic and enteroinvasive Escherichia are the etiological agents of bacterial diarrheas on the territory of the Moscow region. The dynamics of the etiological agents of AEI in the region has been established.     

Biological properties of opportunistic microorganisms isolated from patients with acute intestinal diseases

The comparative study of the incidence of the pathogenicity markers (DNAase, RNAase, phosphatase and hemolytic activity) in shigellae and salmonellae, acknowledged as the causative agents of intestinal infections, and in opportunistic bacteria isolated from the feces of patients with acute intestinal diseases and healthy persons has been made. The study has revealed that DNAase and RNAase activity occurs most frequently in Shigella flexneri, in salmonellae and in opportunistic enterobacteria isolated from the intestinal contents of patients with acute intestinal diseases. In this respect they essentially differ from the same species of opportunistic enterobacteria isolated from healthy persons.     

Neutralization of the Bactericidal Effect of Cocoa Powder on Salmonellae by Casein

Four pre-enrichment media (nutrient broth, lactose broth, reconstituted non-fat dry milk and nutrient broth containing 5% (w/v) casein) were evaluated for the recovery of salmonellae from cocoa powder. Addition of 5% cocoa powder to nutrient broth and to lactose broth proved to be bactericidal to salmonellae. Heat-damaged Salmonella typhimurium were more sensitive than undamaged cells to cocoa powder and agitation increased the bactericidal effect. The bactericidal effects were minimized in pre-enrichment media that contained either 5% casein or nonfat dry milk.     

Inefficient in vitro killing of virulent or nonvirulent Salmonella typhimurium by murine polymorphonuclear neutrophils

The bactericidal capability of murine peritoneal polymorphonuclear neutrophils against virulent and nonvirulent Salmonella typhimurium was examined in an in vitro system. Although preincubation of the bacteria in specific murine antiserum elicited greater chemiluminescence from phagocytizing neutrophils than did incubation in normal murine serum, antiserum did not enhance ingestion, as less than 5% of the challenge was taken up by neutrophils under any of the conditions studied. Nonvirulent salmonellae showed a transient decrease in viable numbers early during in vitro incubation with or without intact neutrophils. Virulent salmonellae, however, were able to multiply without a lag period except when these bacteria were pretreated with antiserum and incubated in association with intact murine neutrophils. Results of these in vitro studies suggest that the murine polymorphonuclear neutrophil and antisalmonella antibody must act together to effect neutrophil-associated bactericidal activity against virulent salmonellae, and thus, that the neutrophil alone does not play a major role in the protection of unvaccinated, sensitive mice from disease caused by S. typhimurium.     

The fibronectin binding of salmonellae studied by a passive hemagglutination method]

The study has shown that the fibronectin-binding properties of different Salmonella species can be tested by means of passive hemagglutination with fibronectin-sensitized formulated sheep red blood cells (SRBC). S. enteritidis and S. typhimurium isolated from foodstuffs, as well as the representatives of the species S. kottbus, S. anatum, S. tshiongwe and S. newlands, are capable of binding fibronectin. Fibronectin-binding properties have also been detected in Shigella sonnei, as well as in eubiotic microorganisms Escherichia coli M 17 and Lactobacillus plantarum. In pH ranging from 6.5 to 7.4 salmonellae, shigellae and E. coli agglutinate fibronectin-sensitized SRBC much more effectively in acidic pH values, while changes in pH have been found to produce no essential influence on the fibronectin-binding capacity of Staphylococcus aureus and lactobacilli.     

Molecular Relationships Among the Salmonelleae

Polynucleotide sequence relatedness studies were carried out to determine the extent of divergence present in members of the tribe Salmonelleae and between salmonellae and other enteric bacteria. Typical Salmonella were 85 to 100% related. Two groups of biochemically atypical Salmonella showed somewhat lower binding to typical salmonellae and to each other. Arizona were 70 to 80% related to salmonellae. Two groups of Arizona were detected. These groups correlated with the presence of monophasic or diphasic flagellar antigens. Salmonella and Arizona were no more related to Citrobacter than to Escherichia coli (45-55%). Relatedness of Salmonella and Arizona to other enterobacteria ranged from 20 to 40% with klebsiellae and shigellae, to 20 to 25% with erwiniae, and to less than 20% with edwardsiellae and Proteus mirabilis.     

Influence of cycloferon on the biological properties of bacterial intracellular pathogens

The influence of cycloferon on some biological properties of pathogenic (Shigella spp., Salmonella spp., Francisella tularensis, Brucella spp.) and opportunistic (Escherichia coli, Staphylococcus spp.) microorganisms has been experimentally determined in vitro. As revealed in these experiments, the preparation used at concentrations under study considerably suppresses the antilysozyme and anticomplementary activity of Gram-negative intracellular parasites (shigellae, salmonellae, F. tularensis, Brucella). These data reveal one of the possible mechanisms of the action of cycloferon on intracellular parasites in acute and chronic bacterial infections.     

Comparison of Media for Isolation of Salmonellae and Shigellae from Fecal Specimens

Five transport media, eight plating media, and three enrichment broth media for the isolation of salmonellae and shigellae were evaluated. Eight laboratories in widely separated regions of the United States participated in this evaluation by submitting 490 fecal specimens in the transport media provided. The results suggest that the newer transport media may not offer any advantage over the use of buffered glycerol-saline in the isolation of these enteric pathogens. Shigellae were best isolated by direct inoculation, whereas salmonellae were isolated in greater numbers after tetrathionate (without Brilliant Green) enrichment with subsequent culturing on the plating medium. The use of a variety of plating media is recommended for the recovery of a larger number of these enteric pathogens.     

Isolation of Salmonellae and Shigellae from an Artificial Mixture of Fecal Bacteria

Numerous selective media, available commercially, act by suppressing "normal" bacterial inhabitants of the intestine while permitting the growth of so-called pathogenic representatives of the family Enterobacteriaceae. This investigation attempts to evaluate the action of Salmonella-Shigella (SS) agar, xylose lysine desoxycholate (XLD) agar, and hektoen enteric (HE) agar. Salmonellae and shigellae, isolated from clinical material, were mixed in various ratios with escherichiae, Klebsiella-Enterobacter-Serratia group bacteria, and members of the tribe Proteeae, also of clinical origin. Several of the mixtures were plated in multiple dilutions on the three media. Stools in preservative were also used for evaluation of the media after the addition of definite numbers of the pathogenic bacteria. Results indicate that SS agar suppresses the shigellae along with the autochthonous members of Enterobacteriaceae. XLD and HE agars readily permit the recovery of shigellae as well as salmonellae. This recovery is not obscured by the higher yield of other species obtained with these media.     

Lysine-Iron Agar in the Detection of Arizona Cultures

A lysine-iron agar is described and recommended for the detection of Arizona strains which ferment lactose rapidly. Black colonies which appear on bismuth sulfite agar should be transferred to the medium. Salmonellae and Arizona cultures produce a distinctive reaction since they are the only recognized groups of enteric bacteria which regularly produce lysine decarboxylase rapidly and form large amounts of hydrogen sulfide. Use of the medium is particularly recommended in the examination of specimens from enteric infections in which shigellae and salmonellae are not detected.     

Cross-Agglutination Reaction in Candida albicans and Enteric Bacilli

Cross-reactivity between Candida albicans and representative Enterobacteriaceae was investigated by agglutination methods. It was observed that anti-Candida serum reacted to certain groups of salmonellae and shigellae. The only antiserum against the enteric bacilli that reacted with Candida was Salmonella C(1). When anti-Candida serum was adsorbed with C. albicans or S. montevideo, all the activity was removed. However, when the anti-Salmonella serum was adsorbed with the antigens, Salmonella adsorbed the whole antibody activity, whereas Candida removed only its corresponding antibody.     

Gibbs free energy in periodic enterobacterial cultures]

The authors present the results of study of the Gibbs' free energy (--delta G) output in periodic cultures of nonpathogenic and enteropathogenic escherichia, shigellae, salmonellae, Proteus, serratia, and klebsiellae in the hydrolysate broth in a special twelve--chamber cultivator. Graphic expression--deltaG = f(N) permitted to reveal differences in the energy characteristics of individual enterobacteria genera and species. It is supposed that such studies could be useful in the choice of redox-indicators during the construction of differential-diagnostic media for the laboratory diagnosis of intestinal infections.     

Antibacterial activity of the lactoperoxidase system against Aeromonas hydrophila in broth, skim milk and ewes' milk

The effect of the lactoperoxidase (LP)-system on Aeromonas hydrophila in broth, skim milk and ewes' milk was investigated. The behaviour of three strains in activated and control substrates was studied at 6 and 28°C. The activated ovine lactoperoxidase was bactericidal for Aer. hydrophila . In the three LP-treated substrates the tested strains were completely inactivated after 2 (28°C) and 24 h (6°C) and then remained undetectable during storage for up to 25 d. The use of the LP-system as a preservative against Aer. hydrophila could be very useful even when adequate cooling facilities are available.