Nisin treatment for inactivation of Salmonella species and other gram-negative bacteria.

Nisin, produced by Lactococcus lactis subsp. lactis, has a broad spectrum of activity against gram-positive bacteria and is generally recognized as safe in the United States for use in selected pasteurized cheese spreads to control the outgrowth and toxin production of Clostridium botulinum. This study evaluated the inhibitory activity of nisin in combination with a chelating agent, disodium EDTA, against several Salmonella species and other selected gram-negative bacteria. After a 1-h exposure to 50 micrograms of nisin per ml and 20 mM disodium EDTA at 37 degrees C, a 3.2- to 6.9-log-cycle reduction in population was observed with the species tested. Treatment with disodium EDTA or nisin alone produced no significant inhibition (less than 1-log-cycle reduction) of the Salmonella and other gram-negative species tested. These results demonstrated that nisin is bactericidal to Salmonella species and that the observed inactivation can be demonstrated in other gram-negative bacteria. Applications involving the simultaneous treatment with nisin and chelating agents that alter the outer membrane may be of value in controlling food-borne salmonellae and other gram-negative bacteria.     

The influence of cations on the permeability of the outer membrane of Salmonella typhimurium and other gram-negative bacteria.

The high sensitivity of rough mutants of Salmonella typhimurium, S. minnesota, and Escherichia coli 08 (i.e. with defects in the carbohydrate core of the lipopolysaccharide) to several antibiotics and to the dye gentian violet could be substantially reduced by the addition of cations (Mg2+, Na+) into the growth medium. One heptoseless mutant of S. typhimurium (chemotype Re) and its isogenic smooth parent strain were studied in more detail. The uptake of gentian violet was about 20% in the smooth strain, about 60% in the Re strain grown without additional cations, but decreased to about 15% in the same strain, when cations had been present during growth. In all cases, almost 50% of the gentian violet taken up by the cells was membrane-bound. The total membranes of the Re strain grown in nutrient broth without additional Mg2+ ions were reduced in the 36K and 34K major outer membrane proteins compared with the smooth strain; when grown with added cations the Re total membranes (and even whole cells) did not revert to the protein pattern of the smooth strain.     

Genetic tools for pseudomonads, rhizobia, and other gram-negative bacteria

Gram-negative bacteria are extraordinarily diverse microorganisms that present a wide variety of characteristics worthy of genetic investigation. For historical reasons, the application of recombinant DNA technology to gram-negative bacteria in general has always lagged behind that of E. coli and its close relatives. However, the past 10 years have seen dramatic advances in the development of new tools and vectors for genetic analysis in non-E. coli hosts. Applications include various kinds of genetic manipulation, conjugation, transposition, site-specific recombination, protein secretion, protein purification, cell suicide, microbial ecology, biodegradation, and plant and animal pathogenicity.     

Expansion of the host range of coliphage P1 and gene transfer from enteric bacteria to other gram-negative bacteria.

The bacterial host range of coliphage P1 was extented by using the heat-inducible phage P1clr100KM. A gene for kanamycin resistance was transferred from Escherichia coli to members of the family Enterobacteriaceae and some other genera of gram-negative bacteria. P1 phage was produced by thermal induction from the lysogens of all these kanamycin-resistant bacteria except some strains.     

Expansion of the host range of coliphage P1 and gene transfer from enteric bacteria to other gram-negative bacteria.

The bacterial host range of coliphage P1 was extented by using the heat-inducible phage P1clr100KM. A gene for kanamycin resistance was transferred from Escherichia coli to members of the family Enterobacteriaceae and some other genera of gram-negative bacteria. P1 phage was produced by thermal induction from the lysogens of all these kanamycin-resistant bacteria except some strains.     

Incidence of Acinetobacter spp. and other gram-negative, oxidase-negative bacteria in fresh and spoiled ground beef.

A total of 1,409 gram-negative bacterial colonies were randomly selected from 19 samples of fresh and spoiled ground beef plated on six media. Only 137 (9.7%) were oxidase negative, and 20 (14.6%) of these were Acinetobacter spp., all of which were recovered from fresh meat samples. The importance of this group in both fresh and spoiled beef is less than is generally believed.     

Expression of the Escherichia coli pfkA gene in Alcaligenes eutrophus and in other gram-negative bacteria.

The Escherichia coli pfkA gene has been cloned in the non-self-transmissible vector pVK101 from hybrid plasmids obtained from the Clarke and Carbon clone bank, resulting in the plasmids pAS300 and pAS100; the latter plasmid also encoded the E. coli tpi gene. These plasmids were transferred by conjugation to mutants of Alcaligenes eutrophus which are unable to grow on fructose and gluconate due to lack of 2-keto-3-deoxy-6-phosphogluconate aldolase activity. These transconjugants recovered the ability to grow on fructose and harbored pAS100 or pAS300. After growth on fructose, the transconjugants contained phosphofructokinase at specific activities between 0.73 and 1.83 U/mg of protein, indicating that the E. coli pfkA gene is readily expressed in A. eutrophus and that the utilization of fructose occurs via the Embden-Meyerhof pathway instead of the Entner-Doudoroff pathway. In contrast, transconjugants of the wild type of A. eutrophus, which are potentially able to catabolize fructose via both pathways, grew at a decreased rate on fructose and during growth on fructose did not stably maintain pAS100 or pAS300. Indications for a glycolytic futile cycling of fructose 6-phosphate and fructose 1,6-bisphosphate are discussed. Plasmid pA 100 was also transferred to 14 different species of gram-negative bacteria. The pfkA gene was expressed in most of these species. In addition, most transconjugants of these strains and of A. eutrophus exhibited higher specific activities of triosephosphate isomerase than did the corresponding parent strains.     

Incidence of Acinetobacter spp. and other gram-negative, oxidase-negative bacteria in fresh and spoiled ground beef

A total of 1,409 gram-negative bacterial colonies were randomly selected from 19 samples of fresh and spoiled ground beef plated on six media. Only 137 (9.7%) were oxidase negative, and 20 (14.6%) of these were Acinetobacter spp., all of which were recovered from fresh meat samples. The importance of this group in both fresh and spoiled beef is less than is generally believed.     

Phospholipase D activity of gram-negative bacteria.

A phospholipase hydrolyzing cardiolipin to phosphatidic acid and phosphatidyl glycerol was characterized in gram-negative bacteria but was absent in preparations of gram-positive bacteria, Saccharomyces cerevisiae, and rat liver mitochondria. In cell-free extracts of Escherichia coli, Salmonella typhimurium, Proteus vulgaris, and Pseudomonase aeruginosa, this cardiolipin-hydrolyzing enzyme had similar pH and Mg2+ requirements and displayed a specificity which excluded phosphatidyl glycerol and phosphatidyl ethanolamine as substrates.     

New plate medium for facilitated differentiation of Salmonella spp. from Proteus spp. and other enteric bacteria

A new agar medium for the differentiation of Salmonella spp. from other members of the family Enterobacteriaceae is described. This medium exploits a novel phenotypic characteristic of Salmonella spp.: the formation of acid from propylene glycol. This characteristic may be used in combination with a chromogenic indicator of beta-galactosidase to differentiate Salmonella spp. from Proteus spp. and the other members of the Enterobacteriaceae. Desoxycholate may be included in the plate medium as an inhibitor of gram-positive organisms. Non-typhi Salmonella spp. yield distinct, bright red colonies on this medium, allowing facilitated identification and unambiguous differentiation from Proteus spp.     

Removal and inactivation of bacteria during alum treatment of a lake.

Flocculation and removal of bacteria were observed during two separate aluminum sulfate (alum) treatments for removal of phosphorus from a eutrophic recreational lake. In addition, die-off and release of bacteria from alum floc were studied in columns under laboratory conditions. Membrane filtration and spread plates were used to determine concentrations of indicator species and total cultivatable bacteria, respectively. During the alum treatment of the lake, 90% of the fecal coliform (FC) population and ca. 70% of the fecal streptococci population were removed from the water column within 72 h. Numbers of FC in the floc on the lake bottom exceeded 2,400/100 ml at 120 h compared with the pretreatment concentration of 30 FC/100 ml. Inactivation of FC in the floc proceeded at a rate of 200 FC/100 ml per 24 h. In a second alum application to the lake, 95% of the total culturable bacterial population was removed from the water column. In a laboratory column study of survival and release rates, over 90% of an Escherichia coli suspension was concentrated in a floc formed at the bottom. E. coli was not released from the floc. The numbers of and survival of E. coli in the floc suggest the probable concentration of other enteric organisms, including pathogens. Thus, the floc poses a potential human health risk if ingested by swimmers or if others use the lake as a potable water source.     

Distribution of insertion sequence ISRm1 in Rhizobium meliloti and other gram-negative bacteria

An internal 0.9 kb segment of Rhizobium meliloti insertion sequence ISRm1 was used as a probe to determine the distribution of ISRm1 in strains of R. meliloti and other Gram-negative bacteria. The insertion sequence was detected in 80% (12/15) of R. meliloti strains from different parts of the world. Its copy number ranged from one to at least eleven. The ISRm1 copies detected showed variation in their internal restriction sites and their degree of homology to the probe. ISRm1 was found in a variety of genomic restriction fragments, and was detected in plasmids, including the nod and exo megaplasmids of R. meliloti. Other rhizobia found to contain ISRm1 were a strain of R. leguminosarum biovar phaseoli and two Rhizobium isolates capable of nodulating both Medicago sativa and Phaseolus vulgaris. It was also found in a diazotrophic soil bacterium isolated from the roots of wetland rice.     

New plate medium for facilitated differentiation of Salmonella spp. from Proteus spp. and other enteric bacteria.

A new agar medium for the differentiation of Salmonella spp. from other members of the family Enterobacteriaceae is described. This medium exploits a novel phenotypic characteristic of Salmonella spp.: the formation of acid from propylene glycol. This characteristic may be used in combination with a chromogenic indicator of beta-galactosidase to differentiate Salmonella spp. from Proteus spp. and the other members of the Enterobacteriaceae. Desoxycholate may be included in the plate medium as an inhibitor of gram-positive organisms. Non-typhi Salmonella spp. yield distinct, bright red colonies on this medium, allowing facilitated identification and unambiguous differentiation from Proteus spp.     

Replication of plasmids in gram-negative bacteria.

Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical data about the initial priming reaction of DNA synthesis exist. Detailed models have been developed for the initiation and regulation of ColE1 replication. For other plasmids, such as pSC101, some hypotheses for priming mechanisms and replication initiation are presented. These hypotheses are based on experimental evidence and speculative comparisons with other systems, e.g., the chromosomal origin of E. coli. In most cases, knowledge concerning plasmid replication is limited to regulation mechanisms. These mechanisms coordinate plasmid replication to the host cell cycle, and they also seem to determine the host range of a plasmid. Most plasmids studied exhibit a narrow host range, limited to E. coli and related bacteria. In contrast, some others, such as the IncP plasmid RK2 and the IncQ plasmid RSF1010, are able to replicate in nearly all gram-negative bacteria. This broad host range may depend on the correct expression of the essential rep genes, which may be mediated by a complex regulatory mechanism (RK2) or by the use of different promoters (RSF1010). Alternatively or additionally, owing to the structure of their origin and/or to different forms of their replication initiation proteins, broad-host-range plasmids may adapt better to the host enzymes that participate in initiation. Furthermore, a broad host range can result when replication initiation is independent of host proteins, as is found in the priming reaction of RSF1010.     

Cloning and transposon vectors derived from satellite bacteriophage P4 for genetic manipulation of Pseudomonas and other gram-negative bacteria.

We developed transposon and cloning shuttle vectors for genetic manipulation of Pseudomonas and other gram-negative bacteria, exploiting the unique properties and the broad host range of the satellite bacteriophage P4. P4::Tn5 AP-1 and P4::Tn5 AP-2 are suicide transposon vectors which have been used for efficient Tn5 mutagenesis in Pseudomonas putida. pKGB2 is a phasmid vector with a cloning capacity of about 7.5 kb; useful unique cloning sites are SacI and SacII in the streptomycin resistance determinant and PvuI and XhoI in the kanamycin resistance determinant. pKGB4 is a cosmid derived from pKGB2 and carries the additional cloning site SmaI in the kanamycin resistance determinant; its cloning capacity is about 18 kb. These vectors and their recombined derivatives were transferred from Escherichia coli to P. putida by transduction and may be used for other bacterial species susceptible to P4 infection.