Selected properties of lactose-fermenting and non-fermenting Salmonella agona strains isolated from specimens from hospitalized infants

The aim of this study was to compare some of the properties of 28 lactose-positive and 28 lactose-negative Salmonella agona strains isolated from faeces of infants hospitalized in the same hospital. Some of biochemical properties, sensitivity to 14 antibiotics and chemotherapeutic agents and sensitivity to bacteriophages used for typing of this Salmonella genus were tested. Results of biochemical examinations revealed that lactose-fermenting strains retain the remaining of Salmonella of subspecies I. Two biochemical features are of particular importance: the ability to ferment lactose on all lactose containing media and a lack of the ability to produce H2S on Kligler medium. These two features differentiate lactose-fermenting strains of Salmonella from non-lactose fermenting ones. Antibiotic sensitivity pattern differed between lactose-positive and lactose-negative strains. Lactose-positive strains showed higher degree of resistance than lactose-negative strains. The differences in resistance were seen in the case of chloramphenicol, doxycycline, gentamicin and tetracycline. Both lactose-positive and lactose-negative strains were sensitive to colistin, neomycin, nitrofurantoin and nalidixic acid. They were resistant to ampicillin, cloxacillin, rifampicin, streptomycin, sulfatiazol and biseptol. Bacteriophage typing revealed that all lactose-negative strains isolated in this study from clinical samples belonged to the same phage pattern V. Lactose-positive strains belonged to two phage types VB and XI. Type VB prevailed.     

Salmonella identification after incubation in the soil

We have tried to study the fate of Salmonella strains in soil. We have looked at their biochemical modifications during their evolution to dormant state and during their reviviscence. The beta galactosidase which is negative in the parent strain became positive after two weeks of cells starvation in soil. Stressed cells became able to produce acetoin. Some stressed cells did not produce the lysine decarboxylase, which is positive in parent cells. These modifications are reversible and depend on cultural conditions. Incubation of stressed cells in nutrient broth for more than four weeks helped them to reverse to normal forms. Simultaneous search for atypical Salmonella was done in dissect sludge of a domestic wastewater treatment plant and in soil irrigated with treated water. Atypical strains of Salmonella are found. We have seen that, after incubation in nutrient broth for more than four weeks, atypical strains characters evolved generally to their parental characters. All modifications of Salmonella in soil samples can make their identification very difficult and perhaps impossible.     

Bacteriophage types represented by lactose-fermenting Salmonella agona strains

Bacteriophage typing was performed on 1911 S. agona, lactose-fermenting strains. These strains were isolated from hospitalised newborns and neonates patients. Out of 1911 strains 98.8% were typable by means of phage set prepared for strains differentiation of Salmonella agona showing typical biochemical properties. It was shown that in 16 provinces from which the strains were obtained in 1983-1985 type V (49.5%) and type XI (25.4%) prevailed. Subtypes VA and VB were distinguished within type V. Altogether 20.3% of strains were classified as belonging to these subtypes. Their lytic reaction was weaker with phages 3, 4, and 9 with the characteristic range of phage type V strains. Among tested strains types I, XIII, and XVI were also represented composing 2, 6, 0, 9, and 0.3% of total number of strains respectively. 1.5% of strains were nontypable and 0.2% showed lytic reactions different from that included in up to now used scheme of typing. It can be concluded that lactose-fermenting S. agona strains show susceptibility to lowered number of phages than typical for Salmonella species strains. It seems that differentiation of this atypical biochemical variant of S. agona with, the use of phage set used up to now may be also usefull in practice as it is the case in respect to strains with typical biochemical properties.     

A PCR-based strategy for simple and rapid identification of rough presumptive Salmonella isolates

The purpose of the present study was to investigate the application of ready-to-go Salmonella PCR tests, based on dry chemistry, for final identification of rough presumptive Salmonella isolates. The results were compared with two different biotyping methods performed at two different laboratories. The sensitivity of the BAX Salmonella PCR test was assessed by testing a total of 80 Salmonella isolates, covering most serogroups, which correctly identified all the Salmonella strains by resulting in one 800-bp band in the sample tubes. The specificity of the PCR was assessed using 20 non-Salmonella strains, which did not result in any DNA band. A total of 32 out of the 36 rough presumptive isolates were positive in the PCR. All but one isolate were also identified as Salmonella by the two biochemical methods. All 80 Salmonella strains were also tested in the two multiplex serogroup tests based on PCR beads. All strains belonging to the serogroups B, C1, C2-C3, and D were grouped correctly. Among the 32 rough presumptive isolates identified, 19 isolates resulted in a band of 882 bp (serogroup B), 11 isolates resulted in a band of 471 bp (serogroup C1), and two isolates showed a band of 720 bp (serogroup D). In conclusion, rough presumptive Salmonella isolates can be conveniently confirmed to the serogroup-level, using the pre-mixed PCR tests. The system can be easily implemented in accredited laboratories with limited experience in molecular biology.     

A novel multiplex PCR assay for Salmonella subspecies identification

Aim:  To develop a novel multiplex polymerase chain reaction (PCR) assay with six primer pairs for Salmonella subspecies identification.
Methods and Results:  Five primer pairs were chosen to detect the genes ( fljB , mdcA , gatD , stn and STM4057) responsible for several phenotypic traits or encoding (sub) species-specific regions. A primer pair for invA was added to simultaneously detect Salmonella . The combination of these primer pairs was expected to give unique results to all subspecies, including Salmonella bongori. The multiplex PCR assay was optimized and evaluated with 53 Salmonella strains representing all S. enterica subspecies, S. bongori and five non- Salmonella strains. The multiplex PCR assay revealed that the genotypes were well correlated with the phenotypes in the Salmonella strains tested. The unique band patterns to their subspecies were generated from 94·3% (50/53) of the Salmonella strains, and no product from other strains by the multiplex PCR assay.
Conclusions:  The multiplex PCR assay we developed was found to be a rapid, specific and easy to perform method compared with traditional biochemical tests for Salmonella subspecies identification, especially for rapid screening of large numbers of samples.
Significance and Impact of the Study:  The assay will be useful for characterizing Salmonella isolates from reptiles, which belong to various subspecies, and therefore add to the scientific understanding of reptile-associated Salmonellosis.     

Biochemical properties of Corynebacterium amycolatum strains

C. amycolatum is the most commonly isolated nonlipophilic species of Corynebacterium from clinical samples. However, the lack of good commercial identification tests in microbiology laboratories causes some difficulties in C. amycolatum diagnostics. We decided to examine biochemical and enzymatic properties of isolated strains and analyze the occurrence of particular biochemical profiles (biotypes). Perhaps it would let improve the identification schemes. 70 strains of C. amycolatum were analyzed. The estimation of biochemical properties consisted of the results of API Coryne and API ZYM tests (bioMérieux), the ability of excreting of protease, esterase, lipase and lecithinase. Analyzed strains had various biochemical and enzymatic properties. Almost all strains fermented glucose (98.6%) and maltose (95.7%) and produced pyrasinamidase (94.3%). All strains produced alkaline phosphatase and phosphohydrolase, and 95.7%--acid phosphatase. Biotypes of particular strains were determined on the biochemical reactions included in the API Coryne tests. In the group of 70 strains 21 profiles were distinguished among which 3100325 biotype (35.7%) was dominant. The lipolysis was defined on Tween 20, Tween 40, Tween 60, Tween 80 medium and with the API ZYM test usage. All strains produced esterase-lipase (esterase C-8), 95.7% of strains-esterase C-4, and 21.4% lipase C-14. Among analyzed strains 18.6% hydrolyzed Tween 20, 14.3% Tween 60, and 1.4% Tween 40. None of these strains demonstrated lipase and lecithinase activity. Difficulties in concerning C. amycolatum as pathogens justify further investigations.     

Vaccine efficacy in spotted wolffish Anarhichas minor: relationship to molecular variation in A-layer protein of atypical Aeromonas salmonicida

Atypical Aeromonas salmonicida strains comprise a heterogeneous group in terms of molecular and phenotypic characteristics. They cause various conditions of ulcer diseases or atypical furunculosis and are being isolated in increasing number from various fish species and geographical areas. Several marine fish species susceptible to atypical A. salmonicida, including spotted wolffish Anarhichas minor O., are now being farmed and new vaccines may be needed. A commercial furunculosis vaccine for salmon is reported to protect wolffish poorly against experimental challenge with atypical A. salmonicida. The protective antigen(s) in furunculosis vaccines is still unclear, but in oil-adjuvanted vaccine for Atlantic salmon Salmo salar L., the surface A-layer was shown to be important for protection. In spotted wolffish, the efficacy of atypical furunculosis vaccines seems to vary with the atypical A. salmonicida strains used as bacterin in the vaccine. In the present study we investigated whether differences in the A-layer protein among atypical strains might be responsible for the observed variation in vaccine efficacy. Atypical A. salmonicida strains from 16 fish species in 11 countries were compared by genome polymorphism analysis using amplified fragment length polymorphism (AFLP) fingerprinting and by comparative sequencing of the vapA genes encoding the A-protein. The A-protein sequences appeared to be highly conserved except for a variable region between Residues 90 to 170. Surprisingly, the grouping of strains based on AFLP- or A-protein sequence similarities was consistent. In addition, serological differences in the A-protein among the strains were demonstrated by an A-protein-specific monoclonal antibody. Vaccines based on atypical A. salmonicida strains possessing genetically and serologically different A-layer proteins were shown to result in significantly different protection in spotted wolffish.     

Selection of methods of detecting lactose-fermenting Salmonella strains and evaluating their usefulness for diagnostic studies

Salmonella rods of subspecies I, lactose-fermenting were first isolated in Poland in 1980. They were isolated from a plus sample taken from a brain abscess of a child. Next strains were isolated from faeces of newborn and hospitalized children. Growth characteristic of colonies of lactose-fermenting Salmonella strains on selective-differentiating media (Mac Conkey's Levine, SS, So?tys) recommended for inoculation of clinical material resembled Escherichia coli. So far these type of colonies were omitted in diagnostic examinations. Lactose-fermenting variants showed on Bismuth sulfate agar "Difco" (WB) typical for Salmonella growth pattern. They grew on this medium after 48 hr of incubation in a form of black, medium sized colonies, with some metallic brilliance and characteristic blackening of the medium undercolonies. Precise knowledge of biochemical properties of lactose-fermenting Salmonella allows to supplement so far used diagnostic scheme with additional tests permitting differentiation of lactose-fermenting variants of Salmonella from the other members of Enterobacteriaceae family. Taking into consideration biochemical variants in diagnostic procedure i.e. lactose-fermenting Salmonella, allowedns to isolate in the years 1983-1985 lactose-positive strains in 1305 out of 2773 (47%) individuals positive for S. agona. In 1987, 246 persons (28.3%) out of 869 with lactose-fermenting Salmonella of various serotypes were simultaneously infected with lactose-negative variant. Lactose-fermenting strains of Salmonella belonged most frequently to the following genera: S. agona, S. enteritidis, S. oranienburg, S. typhimurium, and S. goldcoast. It was found that the modified diagnostic procedure makes possible the isolation and the identification of lactose-positive varians of Salmonella.     

Identification of the enterobacteria isolated from patients by using the Enterotube test system]

The Enterotube test system intended for the accelerated identification of enterobacteria was used to study 319 strains isolated from patients with suppurative complications after surgical operations and traumatic lesions. The test system was found to allow a reliable identification of Enterobacteriaceae having characteristic biochemical properties. It was also found that the kit intended for 10 tests could prove insufficient for the identification of atypical bacteria. The Enterotube test system made it possible to differentiate 95% of enterobacterial strains; in the identification of bacteria by their genus and species and results obtained with the use of the Enterotube test system and common biochemical methods were found to be identical (in 96.4% of tests).     

The biological properties of Salmonella strains isolated from adult patients at different seasons of the year]

Biological properties of Salmonella strains, isolated in different seasons from patients with the corresponding disease of moderate severity, were compared. Their morphological, biochemical, serologic properties, sensitivity to antibiotics, capacity for synthesizing O-antigen, as well as their virulence for experimental animals, have been studied. Seasonal changes in the virulence of Salmonella strains have been established: the strains isolated in autumn have proved to be more virulent than those isolated in winter. In winter the isolation rate of Salmonella strains resistant to the therapeutic doses of antibiotics is significantly higher than in other seasons. In spring and summer Salmonella O-antigen is synthesized more intensively.     

Comparative evaluation of different pairs of DNA repair-deficient and DNA repair-proficient bacterial tester strains for rapid detection of chemical mutagens and carcinogens

The aim of the present study was to evaluate the usefulness of different pairs of DNA repair-deficient and DNA repair-proficient bacterial tester strains in a mutagenicity/carcinogenicity screen, possibly as complements to the Ames test. 70 carcinogenic and non-carcinogenic compounds, representing a variety of chemical structures, were tested for their DNA-damaging effects, using 6 different DNA-repair-deficient bacterial strains. 2 Bacillus subtilis systems, H17/M45 and HLL3g/HJ-15, were used. The susceptibility of Escherichia coli AB1157 was compared with the susceptibility of 4 recombination-deficient mutants, JC5547, JC2921, JC2926 and JC5519. The test compounds were applied onto paper disks (spot test, ST), or incorporated into a top agar layer (agar-incorporation test, AT). The 2 B. subtilis systems were generally found to be more sensitive and reliable than the assays using E coli. The incorporation of the test compounds in the agar increased the sensitivity of the test for polycyclic aromatic hydrocarbons and other poorly water-soluble compounds. Hydrazines and several other highly polar chemicals could be tested more efficiently when applied onto paper disks. About 30% of the test compounds did not induce any growth inhibition and so could not be tested properly. In order to evaluate the ability of these DNA-repair tests to complement the Ames Salmonella mutagenicity test in a genetic toxicology screening program, results from this study were compared with published data both on mutagenicity in the Ames test and on carcinogenicity. 8 carcinogens generally found to be non-mutagenic for Salmonella were tested: 2 showed DNA-damaging properties (mitomycin C, 1,2-dimethylhydrazine), 5 failed to do so (actinomycin D, griseofulvin, thioacetamide, diethylstilbestrol, safrole), and one (thiourea) was not toxic, so that no classification was possible. 2 non-carcinogenic bacterial mutagens were examined; one, sodium azide, was equitoxic for repair-proficient and -deficient strains, while the other, nitrofurantoin, primarily inhibited repair-deficient strains. The DNA-repair tests failed to indicate the mutagenic and carcinogenic properties of acridine orange. Nalidixic acid, a non-mutagenic DNA synthesis inhibitor, damaged bacterial DNA. Apart from the differences summarized above, carcinogenicity was indicated correctly by the Salmonella S9 assay and most sets of DNA-repair-deficient and DNA-repair-proficient tester strains evaluated in this study. Thus, several more carcinogens could be detected by performing the Ames test and the bacterial DNA-repair tests in tandem than by using either test alone. Nevertheless, the use of both bacterial in vitro systems in a battery of short-term tests for mutagenicity/carcinogenicity evaluation is not considered to be ideal, since the Ames test and the pairs of DNA-repair-deficient and DNA-repair-proficient tester strains used had several shortcomings in common under the conditions of this study.     

Identification of Salmonella with the O-1 Bacteriophage

The O-1 bacteriophage test of Cherry et al. (1954) for the presumptive identification of salmonellae in the diagnostic laboratory was investigated. A phage lysate with a titer of 10(12) plaque-forming units per ml was found to be optimal. This preparation lysed 98.2% of Salmonella strains tested, while maintaining its high specificity for salmonellae. Gram-negative organisms other than salmonellae were resistant to the O-1 phage; however, 5.9% of Escherichia coli strains tested were susceptible. The O-1 phage test is a simple, rapid, inexpensive, sensitive, and specific procedure for the identification of salmonellae in the diagnostic laboratory. A presumptive identification is obtained 1 day earlier than with conventional biochemical tests.     

Atypical Aeromonas salmonicida Isolated from Diseased Turbot (Scophtalmus maximus L.)

Atypical Aeromonas salmonicida were isolated from 3 outbreaks of disease among farmed turbot (Scophthalmus maximus L.) in 3 different farms, 1 from Norway (Nl) and 2 from Denmark (DK1 and DK2). In all 3 cases, the incidence of disease and mortality was high and the main characteristic pathological finding was skin ulcers and septicaemia. The isolated bacteria were subjected to a thorough phenotypic and genotypic examination and comparison in the laboratory. All 3 isolates belonged to A. salmonicida but dis-played some very different biochemical properties. However, the 2 Danish strains, DK1 and DK2 had identical ribotypes but different from that of Nl, whereas the plasmid pro-files of DK1 and Nl were identical but different from that of DK2. These observations emphasize the need for an improvement of our understanding of the taxonomy and epi-demiology of atypical A. salmonicida.     

Growth and Metabolism of Lactic Acid Bacteria during and after Malolactic Fermentation of Wines at Different pH

A new medium, called novobiocin-brilliant green-glucose (NBG) agar, was developed for the isolation of Salmonella spp. and evaluated against other conventionally used media including bismuth sulfite, xylose-lysine decarboxylase, brilliant green-sulfa, hektoen enteric, and salmonella-shigella agars. NBG had recovery rates comparable to the other enteric media tested with pure cultures as well as with naturally contaminated amphibian and reptile waters and fecal specimens. However, NBG, hektoen enteric, and salmonella-shigella agars failed to differentiate Salmonella typhi from a fecal specimen even after enrichment in selenite F. Although Citrobacter freundii could grow and resembled salmonellae on NBG, at no time was the recovery of Salmonella spp. colonies jeopardized by the presence of C. freundii in either seeded or naturally contaminated samples. Confirmation rates of typical colonies from NBG agar also compared favorably to the other media tested; however, bismuth sulfite, although selective, was found to have varied differential characteristics for Salmonella spp. As a result, many more colonies had to be picked, which caused bismuth sulfite agar to have the lowest confirmation rate of the media tested. The distinct advantage that NBG agar offers over the conventional method tested, including bismuth sulfite, is the consistent differential reaction of all Salmonella subgroups including biochemically atypical strains. The medium is inexpensive, easy to prepare, and can be stored for at least 2 weeks at 4 degrees C without loss of selective or differential properties.     

Growth and Metabolism of Lactic Acid Bacteria during and after Malolactic Fermentation of Wines at Different pH

A new medium, called novobiocin-brilliant green-glucose (NBG) agar, was developed for the isolation of Salmonella spp. and evaluated against other conventionally used media including bismuth sulfite, xylose-lysine decarboxylase, brilliant green-sulfa, hektoen enteric, and salmonella-shigella agars. NBG had recovery rates comparable to the other enteric media tested with pure cultures as well as with naturally contaminated amphibian and reptile waters and fecal specimens. However, NBG, hektoen enteric, and salmonella-shigella agars failed to differentiate Salmonella typhi from a fecal specimen even after enrichment in selenite F. Although Citrobacter freundii could grow and resembled salmonellae on NBG, at no time was the recovery of Salmonella spp. colonies jeopardized by the presence of C. freundii in either seeded or naturally contaminated samples. Confirmation rates of typical colonies from NBG agar also compared favorably to the other media tested; however, bismuth sulfite, although selective, was found to have varied differential characteristics for Salmonella spp. As a result, many more colonies had to be picked, which caused bismuth sulfite agar to have the lowest confirmation rate of the media tested. The distinct advantage that NBG agar offers over the conventional method tested, including bismuth sulfite, is the consistent differential reaction of all Salmonella subgroups including biochemically atypical strains. The medium is inexpensive, easy to prepare, and can be stored for at least 2 weeks at 4 degrees C without loss of selective or differential properties.     

The lack of mutagenic properties of patulin and patulin adducts formed with cysteine in Salmonella test systems.

The mutagenic properties of patulin and the patulin adducts formed with cysteine were tested with histidine auxotroph Salmonella typhimurium strains as indicator organisms. The tests were performed by microsomal activation and host-mediated assay. Neither patulin nor patulin--cysteine reaction mixture was mutagenic in these test systems.     

Lysogenicity of atypical strains ofSalmonella paratyphi B

Summary Atypical strains ofS. paratyphi B have been described which caused gastroenteritis or were found by chance in healthy perons. These strains possessed atypical natural phages. The late fermentation of d-tartrate by some strains ofS. paratyphi B was examined.     

Characteristics of bacteria in the genus Proteus isolated from patients with sporadic and group intestinal diseases

The biochemical and biological properties of 148 Proteus strains isolated from patients both in sporadic intestinal infections and in a case of group infection in children's hospital was studied. The study revealed that the etiological factor of the group infection was P. mirabilis belonging to rare serovar 48:2. Proteus organisms isolated in sporadic infections belonged to a great number of serovars. No relationship between the isolated serovar and the nosological form of the intestinal disease was established. Among the Proteus strains under study, 82 strains showed atypical biochemical properties in 1 test or more. No correlation between the clinical diagnosis and the occurrence of atypical strains was established.     

Lactic acid bacteria from chicken carcasses with inhibitory activity against Salmonella spp. and Listeria monocytogenes

This study was conducted to isolate psychrotrophic lactic acid bacteria (LAB) from chicken carcasses with inhibitory activity against strains of Salmonella spp. and Listeria monocytogenes. A total of 100 broiler samples were examined for the presence of LAB. Ninety-two LAB isolates that showed antimicrobial effects against Salmonella spp. and L. monocytogenes were further analysed to examine their LAB (Gram-positive, catalase negative, oxidase negative) and psychrotrophic characteristics (ability to grow at 7 °C). Fifty isolates were further selected and identified initially using standard biochemical tests in miniature (Micro-kits API CH 50) and then by sequencing of the 16s-23s rRNA gene boundary region (Intergenic Spacer Region). By molecular identification, these isolates were classified into 5 different LAB species: Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus johnsonii, Pediococcus acidilactici, and Lactobacillus paralimentarius. None of the isolates produced tyramine or histamine.     

Identification of two new intimin types in atypical enteropathogenic Escherichia coli.

Stool specimens of patients with diarrhea or other gastrointestinal alterations who were admitted to Xeral-Calde Hospital (Lugo, Spain) were analyzed for the prevalence of typical and atypical enteropathogenic Escherichia coli (EPEC). Atypical EPEC strains (eae+ bfp-) were detected in 105 (5.2%) of 2015 patients, whereas typical EPEC strains (eae+ bfp+) were identified in only five (0.2%) patients. Atypical EPEC strains were (after Salmonella) the second most frequently recovered enteropathogenic bacteria. In this study, 110 EPEC strains were characterized. The strains belonged to 43 O serogroups and 69 O:H serotypes, including 44 new serotypes not previously reported among human EPEC. However, 29% were of one of three serogroups (O26, O51, and O145) and 33% belonged to eight serotypes (O10:H-, O26:H11, O26:H-, O51:H49, O123:H19, O128:H2, O145:H28, and O145:H-). Only 14 (13%) could be assigned to classical EPEC serotypes. Fifteen intimin types, namely, alpha1 (6 strains), alpha2 (4 strains), beta1 (34 strains), xiR/b2 (6 strains), gamma1 (13 strains), gamma2/q (16 strains), delta/k (5 strains), epsilon1 (9 strains), nuR/e2 (5 strains), zeta (6 strains), iota1 (1 strain), muR/iota2 (1 strain), nuB (1 strain), xiB (1 strain), and o (2 strains), were detected among the 110 EPEC strains, but none of the strains was positive for intimin types mu1, mu2, lambda, or muB. In addition, in atypical EPEC strains of serotypes O10:H-, O84:H-, and O129:H-, two new intimin genes (eae-nuB and eae-o) were identified. These genes showed less than 95% nucleotide sequence identity with existing intimin types. Phylogenetic analysis revealed six groups of closely related intimin genes: (i) alpha1, alpha2, zeta, nuB, and o; (ii) iota1 and muR/iota2; (iii) beta1, xiR/beta2B, delta/beta2O, and kappa; (iv) epsilon1, xiB, eta1,eta2, and nuR/epsilon2; (v) gamma1, muB, gamma2, and theta; and (vi) lambda. These results indicate that atypical EPEC strains belonging to large number of serotypes and with different intimin types might be frequently isolated from human clinical stool samples in Spain.