Mixed Broth Cultures Research Articles

Comparison of Genotypic and Phenotypic Predictions for Heavy Metal Resistance in Salmonella enterica and Escherichia coli

Salmonella enterica and Escherichia coli are two pathogenic bacteria of worldwide importance that can infect the gastrointestinal tract. Contamination in the food supply chain is an area of concern. Animal feed may be supplemented with essential trace elements, which function as nutritional additives to promote growth & health and optimize production. Bacteria have acquired many metal resistance genes to adapt to the exposure of metals. In this study, our objectives were to evaluate in S. enterica and E. coli, the correlation between the resistance genotype and phenotype to certain heavy metals, and the ability of conjugative plasmids to transfer antimicrobial resistance genes (AMRGs) and heavy metal resistance genes (HMRGs). A total of 10 strains, five S. enterica and five E. coli, were used for this study. Minimal inhibitory concentrations (MICs) were determined for heavy metals: copper, silver, arsenic, and tellurite. The tested isolates showed resistance to copper (5/10; 50%), arsenic (7/10; 70%), and silver (9/10; 90%). Cohen’s Kappa statistics were used to analyze genotype to phenotype agreements. Among the 10 strains sampled, the accordance between geno- and phenotypic heavy metal resistance was fair for copper (kappa = 0.4), none to slight for arsenic (kappa = 0.19) and tellurite (kappa = 0), and no agreement for silver (kappa = -0.19). The transfer of HMRGs was determined in a conjugation experiment performed for all five Salmonella strains as donors using mixed broth cultures. Transconjugants were obtained only from the genotypically tellurite-resistant strain PSU-3260, which yielded a transfer frequency of 10⁻³ transconjugants per donor. In such strain, the tellurite-resistant genes reside on an IncHI2-type plasmid that shares high DNA sequence identity with known HMRG-disseminating Salmonella plasmids. Our results indicated no considerable correlation between the geno- and phenotypic resistance towards heavy metals in the sampled S. enterica and E. coli. The necessity of research in this area is supported by the lack of standardized protocols and MIC clinical breakpoints for heavy metals. KEYWORDS: Heavy metal; resistance; Salmonella; E. coli; agriculture; genotype; phenotype; MIC

A Potent Burkholderia Endophyte against Boxwood Blight Caused by Calonectria pseudonaviculata.

Calonectria pseudonaviculata (Cps) poses an increasing threat to boxwood, a major nursery crop and iconic landscape plant worldwide. Here, we report on a potent biocontrol agent that produces small sage green (SSG) colonies on potato dextrose agar. SSG is a bacterial strain recovered from Justin Brouwers boxwood leaves with unusual response to Cps inoculation. Water-soaked symptoms developed on leaves 2 days after inoculation then disappeared a few days later. This endophyte affected several major steps of the boxwood blight disease cycle. SSG at 107 cfu/mL lysed all conidia in mixed broth culture. SSG at 108 cfu/mL reduced blight incidence by >98% when applied one day before or 3 h after boxwood were inoculated with Cps. Its control efficacy decreased with decreasing bacterial concentration to 103 cfu/mL and increasing lead time up to 20 days. When applied on diseased leaf litter under boxwood plants, SSG reduced Cps sporulation and consequently mitigated blight incidence by 90%. SSG was identified as a new member of the Burkholderia cepacia complex with distinct characters from known clinical strains. With these protective, curative, and sanitizing properties, this Burkholderia endophyte offers great promise for sustainable blight management at production and in the landscape.

In vitro inhibitory activity of Bifidobacterium longum BB536 and Lactobacillus rhamnosus HN001 alone or in combination against bacterial and Candida reference strains and clinical isolates

Bifidobacterium longum BB536 and Lactobacillus rhamnosus HN001 are two strains frequently used as probiotic components in food supplements. The decrease of potentially pathogenic gastrointestinal microorganisms is one of their claimed mechanisms. The aim of this study was to investigate their ability, alone or in combination, to inhibit in vitro the growth of Gram-negative, Gram-positive and Candida reference strains and clinical isolates, using different methods.The cell-free supernatants were obtained by centrifugation and filtration from single or mixed broth cultures and the inhibitory activity was tested using both agar-well diffusion and broth microdilution methods. In order to get some preliminary information about the chemical nature of the active metabolites released in the supernatants, the inhibitory activity was investigated after neutralization, heat and proteolytic treatments.The highest inhibitory activity was shown by the untreated supernatant obtained from broth culture of the two probiotic strains, especially against bacterial reference strains and clinical isolates. This supernatant showed inhibitory activity towards Candida species, too. A decreased inhibitory activity was observed for the supernatants obtained from single cultures and after proteolytic treatment, against bacterial reference strains.The study suggests that the combination of B. longum BB536 and L. rhamnosus HN001 could represent a possible alternative against gastrointestinal and urinary pathogens either as prophylaxis or as treatment.

Positive "Surprise" Broth Cultures in Nonunion Surgery Do Not Require Antibiotic Treatment.

The objective of this study was to review the efficacy of a treatment approach for patients with positive intraoperative cultures during fracture nonunion surgery. The authors performed a retrospective case series at a level I trauma center. In this series, 60 patients without preoperative concern for infection were surgically treated for fracture nonunion. The treatment course of patients after fracture nonunion surgery, including culture results, antibiotic administration, and the presence of clinical infection and radiographic union, was studied. Sixty patients underwent fracture nonunion surgery. Twenty-four patients had a positive intraoperative culture. Fourteen patients had only a positive broth culture, 6 had only a positive routine culture, and 4 had positive mixed (routine and broth) cultures. The most common bacteria was coagulase-negative staphylococci, isolated in 19 of 24 patients, and the only isolated organism in 13 of 24 patients. Patients with a positive broth culture were not treated with antibiotics. Four of 10 patients with either a positive routine or mixed culture grown within 3 days of surgery were treated with antibiotics. All patients achieved clinical healing without signs of infection, and all but 2 patients achieved radiographic union at a mean follow-up of approximately 5 years. In the setting of fracture nonunion surgery, patients with only a positive broth culture and those with only a positive routine or mixed cultures that grew in a delayed fashion (>3 days postoperatively) did not require antibiotic treatment to achieve healing. [Orthopedics. 2019; 42(6):e532-e538.].

Providencia stuartii with VIM-1 metallo-β-lactamase

Providencia stuartii with VIM-1 metallo-β-lactamase

Frequency-dependent advantages of plasmid carriage by Pseudomonas in homogeneous and spatially structured environments

The conditions promoting the persistence of a plasmid carrying a trait that may be mutually beneficial to other cells in its vicinity were studied in structured and unstructured environments. A large plasmid encoding mercury resistance in Pseudomonas fluorescens was used, and the mercury concentration allowing invasion from rare for both plasmid-bearing and plasmid-free cells was determined for different initial inoculum densities in batch-culture structured (filter surface) and unstructured (mixed broth) environments. A range of mercury concentrations were found where both cell types could coexist, the regions being relatively similar in the two types of environment although density-dependent in the unstructured environment. The coexistence is explained in terms of frequency-dependent selection of the mutually beneficial mercury resistance trait, and the dynamics of bacterial growth under batch culture conditions. However, the region of coexistence was complicated by conjugation which increased plasmid spread in the mixed broth culture but not the structured environment.

Role of Quorum Sensing and Antimicrobial Component Production by Serratia plymuthica in Formation of Biofilms, Including Mixed Biofilms with Escherichia coli

We have previously characterized the N-acyl-l-homoserine lactone-based quorum-sensing system of the biofilm isolate Serratia plymuthica RVH1. Here we investigated the role of quorum sensing and of quorum-sensing-dependent production of an antimicrobial compound (AC) on biofilm formation by RVH1 and on the cocultivation of RVH1 and Escherichia coli in planktonic cultures or in biofilms. Biofilm formation of S. plymuthica was not affected by the knockout of splI or splR, the S. plymuthica homologs of the luxI or luxR quorum-sensing gene, respectively, or by the knockout of AC production. E. coli grew well in mixed broth culture with RVH1 until the latter reached 8.5 to 9.5 log CFU/ml, after which the E. coli colony counts steeply declined. In comparison, only a very small decline occurred in cocultures with the S. plymuthica AC-deficient and splI mutants. Complementation with exogenous N-hexanoyl-l-homoserine lactone rescued the wild-type phenotype of the splI mutant. The splR knockout mutant also induced a steep decline of E. coli, consistent with its proposed function as a repressor of quorum-sensing-regulated genes. The numbers of E. coli in 3-day-old mixed biofilms followed a similar pattern, being higher with S. plymuthica deficient in SplI or AC production than with wild-type S. plymuthica, the splR mutant, or the splI mutant in the presence of N-hexanoyl-l-homoserine lactone. Confocal laser scanning microscopic analysis of mixed biofilms established with strains producing different fluorescent proteins showed that E. coli microcolonies were less developed in the presence of RVH1 than in the presence of the AC-deficient mutant.

Detection of Mycobacterium tuberculosis in mixed broth cultures using DNA probes.

OBJECTIVE: To investigate the ability of a commercial acridinium ester-labeled DNA probe (AccuProbe, Gen-Probe Inc., USA) to detect the presence of Mycobacterium tuberculosis complex in a mixed culture with Mycobacterium avium complex. METHODS: The density of organisms required to produce a positive result for Mycobacterium tuberculosis complex alone in broth culture was compared with the density required to produce a positive result in the presence of Mycobacterium avium complex. RESULTS: A threshold density of 1.5 x 106 CFU/mL was required for detection of Mycobacterium tuberculosis and this threshold remained unaltered in the presence of Mycobacterium avium complex. The presence of Mycobacterium avium complex had no effect on detection of Mycobacterium tuberculosis in a mixed broth culture incubated and probed over a 21-day period. CONCLUSIONS: The findings of the study suggest that the presence of Mycobacterium avium complex has no effect on the detection of Mycobacterium tuberculosis and that the Accuprobe test is potentially capable of detecting a dual infection with organisms of both complexes.

Analysis of competition in soil among 2,4-dichlorophenoxyacetic acid-degrading bacteria

Competition among indigenous and inoculated 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was studied in a native Kansas prairie soil following 2,4-D additions. The soil was inoculated with four different 2,4-D-degrading strains at densities of 10(3) cells per g of soil; the organisms used were Pseudomonas cepacia DBO1(pJP4) and three Michigan soil isolates, strain 745, Sphingomonas paucimobilis 1443, and Pseudomonas pickettii 712. Following 2,4-D additions, total soil DNA was extracted and analyzed on Southern blots by using a tfdA gene probe which detected three of the strains and another probe that detected the fourth strain, S. paucimobilis 1443, which belongs to a different class of 2,4-D degraders. P. cepacia DBO1(pJP4), a constructed strain, outcompeted the other added strains and the indigenous 2,4-D-degrading populations. The S. paucimobilis population was the secondary dominant population, and strain 745 and P. pickettii were not detected. Relative fitness coefficients determined in axenic broth cultures predicted the outcome of competition in soil for some but not all strains. Lag time was shown to be a principal determinant of competitiveness among the strains, but the lag times were significantly reduced in mixed broth cultures, which changed the competitive outcome. Plasmids containing the genes for the 2,4-D pathway were important determinants of competitiveness since plasmid pKA4 in P. cepacia DBO1 resulted in the slower growth characteristic of its original host, P. pickettii, rather than the rapid growth observed when this strain harbors pJP4.

Immunological methods in food microbiology

Microbial contamination of foods can now be determined using, unmunochemical methods based on the principle of antibody and antigen interaction. Assay methods in use include radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA), affinity chromatography, immunofluorescence and agglutination. Such methods have been applied for the detection of microbial metabolites, e.g. mycotoxins which can now be determined at levels as low as a few parts per billion (ppb = μg kg −1) to be present in a food sample such as peanuts in a matter of minutes using immunological methods. Bacterial enterotoxins have long been detected in foods using immunoassays: however, recent results of new technologies have allowed for the routine analysis of suspected contaminated foods using simple methods. The presence of viable bacteria such as Salmonella in foods has previously only been achievable after isolation of the bacterium. Now, however, with the advent of more specific and sensitive methods such as the sandwich ELISA and latex agglutination, it is possible to establish the presence or absence of these pathogenic bacteria from a mixed broth culture of the food sample several days prior to traditional testing methods. Thus, when compared with conventional methods immunoassays offer similar detection limits and confidence levels. Furthermore, they simplify sample preparation procedures in relation to extraction of metabolites and growth of micro-organisms.

Transferable plasmid-mediated antibiotic resistance in Bacteroides.

Plasmid-mediated resistance to chloramphenicol (Chlr), erythromycin (Eryr), tetracycline (Tetr) and clindamycin (Clindr) was transferred from three clinical isolates of Bacteroides fragilis and one faecal isolate of B. thetaiotaomicron to strains of B. fragilis, B. distasonis and Escherichia coli, and subsequently to B. fragilis and E. coli second-and third-stage recipients in series. Successful transfer was achieved by membrane-filter and centrifugation techniques that provide stable cell-to-cell contact but not by simple mixed broth culture. Chlr Eryr Tetr and Clindr Eryr were transferred at high frequency (1.9 x 10(-3)-1.8 x 10(-4)) but Tetr was transferred at low frequencies (1-1.6 x 10(-6)). Segregation of resistance markers was observed with selection for Tetr when donors were Chlr Eryr Tetr and Chlr Tetr. All transcipients were identical with the parent recipient strains but had the resistance markers of the donor strains. Resistance to antibiotics other than tetracycline was cured by growth with subinhibitory concentrations of aminoacridines and ethidium bromide for 24 h; cure of solitary Tetr required longer incubation (21 days). Identical plasmid DNA bands were demonstrated by agarose-gel electrophoresis in all the donor and corresponding transcipient strains but plasmids were not found in the recipient strains or in strains cured of resistance. Plasmid-mediated transferable antiobiotic resistance in Bacteroides spp. may compromise the treatment of infections and may provide a reservoir of antibiotic resistance in the intestinal flora.

The anaerobically cultured cecal flora of adult fowls that protects chickens from Salmonella infections.

In this work we have analyzed the bacterial composition of anaerobically cultured cecal contents (mixed broth culture) of adult fowls previously shown able to protect 1 day old chickens from oral Salmonella infections. All four cultures studied in this paper gave complete protection against Salmonella infantis when used undiluted or in the 10(-2) dilution and at least some protection in the 10(-4) dilution. The total aerobic as well as anaerobic counts on the nonselective medium used (VLMH) were of the order of 10(8) viable organisms/ml indicating that the mixed broth culture consisted predominantly of facultative organisms. From a total of 239 colonies isolated on the basis of colony morphology 66 were obligate anaerobes and 173 facultative anaerobic or microaerophilic species. Isolates were selected from each medium. They were grouped and tentatively classified on the basis of their ability to grow on selective media, their colony and cell morphology. Gram stain and products formed from glucose fermentation (obligate anaerobes). Further characterization was performed using conventional carbohydrate fermentation and biochemical tests. However only a minor fraction of the anaerobic isolates could be identified as being identical to known species. The most numerous species were E. coli and various Lactobacilli which were still found in the highest dilution (10(-8)) of the broths. Fecal streptococci were the next most frequent, isolated at the 10(-7) dilution. The obligate anaerobes isolated included Gram-positive cocci and Gram-positive and -negative rods tentatively classified as Eubacteria, Propionibacteria, Clostridia, Fusobacteria and Bacteroides. A large number of them were isolated from the 10(-6) dilution and some species of Eubacteria and Clostridia could only be recovered from the 10(-3) or 10(-4) dilutions.

Method for Rapid Detection of Group B Streptococci by Coagglutination

A quick and reliable technique for the identification of group B streptococci has been developed. The method requires no elaborate equipment or expensive reagents and can be used to detect the group B organisms in mixed broth cultures or to identify suspect colonies selected from agar plates. The method is a coagglutination technique in which 1 drop of specifically sensitized protein A-containing Staphylococcus aureus is mixed with 1 drop of supernatant of an actively growing culture. The soluble group-specific carbohydrate substance of the group B streptococci reacts with the staph particles to produce agglutination that is macroscopically readable. One colony of group B streptococci taken from an agar plate and inoculated into Todd-Hewitt broth will give a positive reaction within 6 h of incubation; with a larger inoculum, the positive reaction occurs within a shorter period. The method was applied for detection of group B streptococci in mixed broth cultures. In laboratory studies involving random mixtures of organisms, 59.3% of positive cultures were detected within the first 8 h of incubation, and 71.7% were found within 24 h. In clinical studies with mixed broth cultures grown directly from vaginal swabs, 78.6% of the positive cultures were detected within the first 8 h of incubation, and 92.9% were found within 24 h.

Bactericidal substance produced by Haemophilus influenzae b.

During bacteriophage studies on Haemophilus influenzer, it was observed that encapsulated type b and unencapsulated Rb strains released a bactericidal substance acitve against types a, c, d, e, and f H. influenzae, non-typable H. influenzae strains, other Haemophilus species, and certain members of the Enterobacteriaceae. The bactericidal activity was assayed by a plaque test utilizing an Rd strain as an indicator lawn and was also demonstrated in mixed broth cultures of a producer strain and an indicator strain. Immediately lysis of sensitive bacteria by the factor was not evident. The factor is sensitive to trypsin but resistant to deoxyribonuclease, treatment with 2-mercaptoethanol, lipase, alpha-amylase, and heating in a 100 degrees C water bath for 20 min. The activity is not dependent upon increased Ca2+ or Mg2+ concentration as is necessary for HP1C1 and S2 phage propagation. The bactericidal factor is not pelleted by high-speed centrifugation at 150,000 X g for 6 h. Treatment with ultraviolet light or mitomycin C does not result in observable phage, phage-like particles, or increased bactericidal activity. T-HE BACTERICIDAL FACTOR IS NOT A TYPICAL SMALL MOLECULAR WEIGHT "COLICIN-LIKE" BACTERiocin in that it is not inducible, has a wider range of activity, and does not kill by "single-hit" kinetics. On preliminary characterization, it is a thermostable protein toxic to certain bacterial strains.

Human salivary glycosidases.

SummaryThe activity of several glycosidases—neuraminidase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, β-N-acetylglucosaminidase, α-mannosidase, β-glucuronidase, and α-L-fucosidase—was assayed in whole, parotid, and submaxillary–sublingual human saliva as well as in mixed cultures of oral microflora. All of these enzymes were present in whole saliva and, with the exception of α-mannosidase, were also present in mixed broth cultures of oral organisms from the same subjects. Only α-L-fucosidase and β-N-acetylglucosaminidase were found in sterile parotid and submaxillary–sublingual saliva. The latter had peaks of optimal pH activity that differed from those of the corresponding bacterial enzymes recovered from the same subject. Since salivary glycosidases can degrade salivary mucins they may be capable, under certain conditions, of attacking the surface layer of mucus in the stomach.

THE NATURE OF INHIBITORY ACTIVITY BY STAPHYLOCOCCUS AUREUS TYPE 71.

SUMMARY: Antibiotic production in liquid media by Staphylococcus aureus strains of bacteriophage ‘type 71’ was poor as measured by cup assays against susceptible corynebacteria. Aerobic incubation in freshly prepared tryptic-digest broth containing a fermentable carbohydrate and adjusted to pH 7.8 gave the greatest yields. The antibiotic material was not obtained in a pure state, but was concentrated by evaporation of crude solutions. These were prepared by adding trichloroacetic acid to broth cultures and centrifuging to remove the organisms. The antibiotic was stable and relatively heat-resistant under acid conditions, but was rapidly destroyed when alkaline; it diffused slowly through cellophan, was adsorbed by charcoal, and was inactivated by trypsin but not by pepsin. These properties suggest that it may be protein or polypeptide in nature. Inhibitory activity by type 71 cocci was shown in mixed broth cultures against other non-inhibitory strains of Staphylococcus aureus, and was similar to that previously observed on solid media.

Transmissible drug resistance in an epidemic strain of Salmonella typhimurium.

Among 309 cultures ofSalmonella typhimurium, phage-type 27, fifteen, isolated from eight patients, were found to be resistant to the three drugs, streptomycin, tetracycline and sulphathiazole. This triple resistance could be transferred by growth in mixed broth culture to a strain ofSkigella sonneiand back again to sensitive cultures ofS. typhimurium. In whole cultures the resistance was stable, but spontaneous loss could be demonstrated in a small proportion of the organisms in such cultures. No elimination of resistance was demonstrated after treatment with acriflavine. Resemblances to the multiple drug resistance in enteric bacteria reported from Japan are noted.The author is most grateful to Dr E. S. Anderson, Director of the Enteric Reference Laboratory, Colindale, N.W. 9, for phage-typing the cultures ofSalmonella typhimurium, and to Dr K. Patricia Carpenter, Director of the Dysentery Reference Laboratory, for supplying cultures ofSh. sonneiwhich were essential for the experimental work.

Observations on a transmissible agent determining sexual differentiation in Bacterium coli

SUMMARY: Analysis of a pair of Bacterium coli K-12 mutants which had ceased to show genetic recombination after storage, implicated mutant 58–161, which had previously behaved as a gene donor strain, as the infertile parent. Infertile 58–161 failed to display recombination when crossed with a gene acceptor strain (W-677) but was able to mate successfully with wild-type K-12 and prototrophic recombinant donor strains, i.e. it had become a gene acceptor. The terms ‘F + ’ and ‘F − ’ have been adopted (after Lederberg, Cavalli & Lederberg, 1952) to denote donor and acceptor strains respectively. Growth of either 58–161/F − or W-677/F − in mixed broth culture with 58–161/F + resulted in the conversion to F + of up to 75 % of re-isolated colonies of the initially F − strain. F − strains converted to F + by strains of dissimilar genotype showed no phenotypic alteration and, therefore, were not recombinants. Washed, mixed cultures on minimal agar yielded an F + conversion rate of only 3·6%, while 100 % recombinants were F + under the same conditions. The F + agent could be transmitted serially through F − strains and was not filterable. While F − × F − crosses were sterile, F + × F − crosses showed maximum fertility. F + × F + crosses were c. 1020 times less productive than F + × F −. The F + agent had a determining effect on the phenotype of recombinants. Thus, when the F + F − relationship was reversed in F + × F − crosses between the same pair of mutants, almost all recombinants which did not show new patterns of unselected marker characters had the phenotype of the F − parent. Among recombinants from F + × F + crosses, the phenotypes of both parents were represented though not always equally. This effect of F + on the phenotype of segregants invalidates much of the evidence for genetic linkage in K-12. Reversal of F + potential in otherwise similar crosses also had a marked effect on the efficiency of prototroph formation on minimal agar supplemented with various growth factors required by one of the parent auxotrophs. A tentative theory of the mechanism of recombination is presented on the basis of this and previous work. This supposes that F + is a non-lytic infectious agent, harboured by F + cells and absent from F − cells, which becomes effectively associated with a part (or parts) of the chromosomes of a small proportion of the cells it inhabits. The F + agent thus acts as a gene carrier in the transfer of genetic material from F + to F − cells.