Resistance to antibiotics in microorganisms predates the use of these drugs. This Review examines why antibiotic resistance is inevitable and where it originates from.

The antibiotic resistome: the nexus of chemical and genetic diversity

The field of probiosis has emerged as a new science with applications in farming and aqaculture as alternatives to antibiotics as well as prophylactics in humans. Probiotics are being developed commercially for both human use, primarily as novel foods or dietary supplements, and in animal feeds for the prevention of gastrointestinal infections, with extensive use in the poultry and aquaculture industries. The impending ban of antibiotics in animal feed, the current concern over the spread of antibiotic resistance genes, the failure to identify new antibiotics and the inherent problems with developing new vaccines make a compelling case for developing alternative prophylactics. Among the large number of probiotic products in use today are bacterial spore formers, mostly of the genus Bacillus. Used primarily in their spore form, these products have been shown to prevent gastrointestinal disorders and the diversity of species used and their applications are astonishing. Understanding the nature of this probiotic effect is complicated, not only because of the complexities of understanding the microbial interactions that occur within the gastrointestinal tract (GIT), but also because Bacillus species are considered allochthonous microorganisms. This review summarizes the commercial applications of Bacillus probiotics. A case will be made that many Bacillus species should not be considered allochthonous microorganisms but, instead, ones that have a bimodal life cycle of growth and sporulation in the environment as well as within the GIT. Specific mechanisms for how Bacillus species can inhibit gastrointestinal infections will be covered, including immunomodulation and the synthesis of antimicrobials. Finally, the safety and licensing issues that affect the use of Bacillus species for commercial development will be summarized, together with evidence showing the growing need to evaluate the safety of individual Bacillus strains as well as species on a case by case by basis.

The use of bacterial spore formers as probiotics

The first vancomycin-resistant clinical isolates of Enterococcus species were reported in Europe in 1988. Similar strains were later detected in hospitals on the East Coast of the United States. Since then, vancomycin-resistant enterococci have spread with unexpected rapidity and are now encountered in hospitals in most countries. This article reviews the mode of action and the mechanism of bacterial resistance to glycopeptides, as exemplified by the VanA type, which is mediated by transposon Tn1546 and is widely spread in enterococci. The diversity, regulation, evolution, and recent dissemination of methicillin-resistant Staphylococcus aureus are then discussed.

Vancomycin resistance in gram-positive cocci.

Enterococci are best known as antibiotic-resistant opportunistic pathogens that are commonly recovered from patients who have received multiple courses of antibiotics and have been hospitalized for prolonged periods.1–6 These organisms were well established as a cause of endocarditis and urinary tract infections by the early 1900s,3 and members of the species Enterococcus faecalis were known to be a common cause of nosocomial infections by the early 1980s.3,7–9 The emergence of enterococci with resistance to vancomycin, seen predominantly in the species E. faecium, has been followed by an increase in the frequency with which this species is recovered. . . .

Vancomycin-resistant enterococcal infections

As the decade progresses this prediction is rapidly being realized, and microbial enzymes are becoming increasingly important in such diverse fields as medicine, brewing, and timber preservation. The genus Bacillus has played a major role in this development as evidenced by the distribution of the papers read at the Fifth International Fermentation Symposium, 1976 (64). Of 23 papers in the session devoted to "Microbial Enzymes of Industrial Interest" no less than ten were concerned with enzymes from bacilli. Reasons for the predominance of these bacteria in this area of study are several. First, they comprise a group of chemoorganotrophs that can be easily maintained and cultivated and yet are markedly heterogeneous in character. Psychrophiles, mesophiles, and thermophiles, in addition to alkalophilic, neutrophilic, and acidophilic species are well represented. Furthermore, virtually all 48 species of the genus listed in Bergey's Manual ofDeterminative Bacteriology (92) secrete a variety of soluble extracellular enzymes, which reflects the diversity of the parental habitats. Amylases that can liquefy starch under pressure at 11000 (194) and proteases that are stable and active at pH 12.0 (6) are extreme examples of enzyme adaption. This article will attempt to review the recent literature concerned with the characterization and properties of the exoenzymes synthesized by the bacilli and the control and mechanisms of their synthesis. It is restricted to this genus because the commercial importance of extracellular enzymes and academic interest in the process of sporulation have prompted a considerable amount of research into this general area. Nevertheless, in the final section I have attempted to equate our present knowledge of exoenzyme synthesis in procaryotes other than

Extracellular enzyme synthesis in the genus Bacillus.

The genetic relatedness of 62 strains of Bacillus sphaericus, 4 strains of B. pasteurii, 6 strains of B. brevis, and 1 strain each of B. globisporus and B. aminovorans was examined by deoxyribonucleic acid (DNA) homology. Five homology groups were identified, and they contained all but 12 of the B. sphaericus strains. Intergroup homologies ranged from about 20 to 40% except for groups III and IV, which had 50 to 60% intergroup homology. Although a few strains within some groups had 63 to 69% homology to the reference strain, most strains were in the 70 to 90% range. Homology group I contained the type of strain of B. sphaericus, ATCC 14577, and three other strains. Homology group II was subdivided into groups IIA and IIB, the former containing all of the strains found to be pathogenic for mosquito larvae. Although none of the physiological characteristics or combination of characteristics examined allowed the complete differentiation of strains into the appropriate homology groups, the strains of group I could be distinguished from those of group IIA-IIB. The strains of group IIA could be distinguished from all others by mosquito pathogenicity. The B. pasteurii strains formed a distinct homology group, although the guanine plus cystosine contents of their DNAs fell within the range of 34 to 37 mol% found for the 45 strains of B. sphaericus examined for this property. The B. brevis strains possessed a higher guanine plus cystosine content, and their DNAs were unrelated to the DNAs of the B. sphaericus reference strains tested.

Deoxyribonucleic Acid Homologies Among Strains of Bacillus sphaericus

Bacillus sphaericus 2297, growing from a boiled, relatively nontoxic spore inoculum, increased about 30-fold in toxicity for mosquito larvae during early exponential growth but showed an approximately 1,000-fold toxicity increase during the late-exponential phase, as spores began to appear in the culture. The development of spores in the bacterial cells was accompanied by the formation of parasporal crystals. These parasporal crystals appeared during stage III as the forespore septum engulfed the incipient forespore. The paraspores were separated from the forespores by a branch of the exosporium across the cell. Measurements of the parasporal substructure revealed a 6.3-nm distance between the striations. When spores and paraspores were fed to mosquito larvae and the larvae were fixed 15 min after feeding, it was found that the spores remained relatively unchanged but that the matrix of the paraspores was dissolved. After dissolution of the paraspore matrix, a meshlike envelope remained which retained the paraspore shape and which was often in contact with the cross-cell portion of the exosporium. The parasporal crystals may be a source of the mosquito larval toxin in this strain of B. sphaericus, but proof will require their isolation from other cellular components.

Ultrastructural Analysis of Spores and Parasporal Crystals Formed by Bacillus sphaericus 2297

Almost complete 16S rRNA gene sequences were generated for the type strains of the obligate insect pathogens Bacillus lentimorbus and Bacillus popilliae and a second strain of Bacillus popilliae (NRRL B-4081) received as ‘ Bacillus popilliae var. melolonthae’. A phylogenetic tree was constructed which grouped these strains into a well defined subcluster within the genus Paenibacillus. Bacillus popilliae NRRL B-4081 occupied an intermediate position between the type strains of Bacillus lentimorbus and Bacillus popilliae but with a marked clustering to the latter. The phylogenetic assignment of these strains to Paenibacillus is in contrast to earlier studies which placed these bacteria in the genus Bacillus, close to Bacillus subtilis. Indeed, the rRNA sequences generated in this study share less than 88% similarity to the deposited sequences for Bacillus popilliae ATCC 14706Tand Bacillus lentimorbus ATCC 14707T. The results obtained by using different tree algorithms, bootstrap analysis, branch lengths and verification by signature nucleotide analysis supported the reclassification of these species in the genus Paenibacillus as Paenibacillus lentimorbus comb. nov. and Paenibacillus popilliae comb. nov.

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Transfer of Bacillus lentimorbus and Bacillus popilliae to the genus Paenibacillus with emended descriptions of Paenibacillus lentimorbus comb. nov. and Paenibacillus popilliae comb. nov.

We have previously identified, in Paenibacillus popilliae, a 708-bp sequence which has homology to the sequence of the enterococcal vanA gene. We have performed further studies revealing five genes encoding homologues of VanY, VanZ, VanH, VanA, and VanX in P. popilliae. The predicted amino acid sequences are similar to those in VanA vancomycin-resistant enterococci: 61% identity for VanY, 21% for VanZ, 74% for VanH, 77% for VanA, and 79% for VanX. The genes in P. popilliae may have been a precursor to or have had ancestral genes in common with vancomycin resistance genes in enterococci. The use of P. popilliae biopesticidal preparations in agricultural practice may have an impact on bacterial resistance in human pathogens.

The Biopesticide Paenibacillus popilliae Has a Vancomycin Resistance Gene Cluster Homologous to the Enterococcal VanA Vancomycin Resistance Gene Cluster

Bacillus thuringiensis var. kurstaki was shown to produce an extracellular, metal chelator-sensitive protease during the early stages of sporulation. Protease production in nutrient broth was dependent upon supplementation with Mn2+ or Ca2+. The addition of Ca2+ was required for enzyme stabilization. Protease production occurred in nutrient broth supplemented with 7 × 10-3 M Ca2+, 5 × 10-4 M Mn2+, and 10-3 M Mg2+. The protease had optimum activity in the pH range 6.5 to 7.5. It was inhibited by chelating agents but not by a serine protease inhibitor. The culture supernatant and the partially purified protease lacked esterase activity. Partial purification of the enzyme (92.3 ×) by (NH4)2SO4 fractionation and starch adsorption yielded an enzyme whose molecular weight was estimated to be 37,500 by acrylamide gel-sodium dodecyl sulfate electrophoresis or 40,800 by sucrose density gradient centrifugation. In the presence of Ca2+, the partially purified enzyme retained 78% of its activity after heating at 70 C for 10 min but only 8% of its activity after heating at 80 C for 10 min.

Allan A. Yousten

Papers

Deoxyribonucleic Acid Homologies Among Strains of Bacillus sphaericus

Ultrastructural Analysis of Spores and Parasporal Crystals Formed by Bacillus sphaericus 2297

Transfer of Bacillus lentimorbus and Bacillus popilliae to the genus Paenibacillus with emended descriptions of Paenibacillus lentimorbus comb. nov. and Paenibacillus popilliae comb. nov.

The Biopesticide Paenibacillus popilliae Has a Vancomycin Resistance Gene Cluster Homologous to the Enterococcal VanA Vancomycin Resistance Gene Cluster

Metalloprotease from Bacillus thuringiensis