Effect of biotin deficiency on some properties of Staphylococcus aureus isolates from humans and other animals.

Staphylococcus aureus isolates from humans and other animals were grown in biotin assay medium containing 12 mug of biotin per liter and compared to isolates from the same sources grown concurrently in medium containing adequate biotin. The two cultures were tested for production of coagulase, phosphatase, and fibrinolysin enzymes and for responses to various antimicrobial agents and bacteriophages. Organisms grown in biotin-deficient medium produced less phosphatase; coagulase and fibrinolytic activity was reduced, and they were more susceptible to antimicrobial agents than were normal organisms, but phage susceptibility was not greatly affected.     

Serotyping of Campylobacter jejuni by slide agglutination based on heat-labile antigenic factors.

A serotyping scheme for Campylobacter jejuni was developed based on slide agglutination of live bacteria with whole cell antisera absorbed with homologous heated and heterologous unheated cross-reactive antigens. Among 815 isolates from human and nonhuman sources, 21 serogroups were recognized. Of the 615 isolates from human cases of gastroenteritis, 529 (86%) were typable; 455 strains agglutinated in 20 single antisera, whereas 74 isolates agglutinated in various pairs of antisera, allowing subdivision of some main serogroups into subserogroups. Of the 200 isolates of C. jejuni from nonhuman sources (chicken, swine, etc.), 166 (83%) were typable, 145 cultures agglutinated in various single antisera, and 21 strains agglutinated with different pairs of antisera. Among isolates from all sources, 8 serogroups (1, 2, 4, 5, 7, 8, 9, and 11) were encountered most frequently. Serogroups 1, 2, 4, 5, 7, 9, and 11 were most common among human isolates; the majority of the chicken and all of the swine isolates belonged to the same serogroups identified from human cases. Very good serological correlation was obtained in 20 family outbreaks and 4 community outbreaks.     

Differentiation of Salmonella serovar infantis isolates from human and animal sources by fingerprinting IS200 and 16S rrn loci.

We genotyped Salmonella serovar infantis (referred to as S. infantis), which is the most widespread serovar among animals and the third most common cause of human salmonellosis in Finland. Molecular fingerprinting of the 16S rrn locus and the Salmonella-specific insertion sequence IS200 was used to type the 131 isolates originating from the main sources of S. infantis infection. The number of IS200 elements in S. infantis varied from zero to seven; three or more copies were present in 97% of the isolates, and 71% had four copies. There were four conserved chromosomal positions of IS200, which allowed us to group the isolates into three major clonal groups. We defined 11 unique IS200 profiles and five different ribotypes which, in combination, generated 15 genotypes highly restricted to the infection sources: 8 genotypes were typical of isolates from broiler chickens and cattle and seven genotypes were typical of isolates from humans. The eight genotypes of isolates from chickens represented two clonal groups which were differentially associated with chicken-producing companies. The typing scheme allows efficient discrimination between isolates from various infection sources and within sources and, therefore, provides a unique molecular tool for use in the study of the epidemiology of S. infantis infection.     

The potential of bacteriocin typing in the study of Clostridium perfringens food poisoning.

A range of 49 bacteriocins was used to type 311 strains of Clostridium perfringens isolated from food poisoning outbreaks. Strains of same serotype within an outbreak showed similar patterns of susceptibility to bacteriocins, whereas strains of different serotype isolated from different sources produced many variations in bacteriocin susceptibility patterns. The 311 strains, along with isolates from a wide range of sources were screened for their ability to produce bacteriocins. A much greater proportion of the strains from food poisoning outbreaks was bacteriocinogenic than were isolates from human and animal infections, various foods and the environment.     

Characterization of Coccidioides immitis isolates by restriction fragment length polymorphisms.

The marked increase in the number of cases of coccidioidomycosis in California in 1992 led to a study of isolates from various patients and environmental sources by restriction fragment length polymorphism (RFLP) analysis. Of 15 different isolates, most of the isolates (13 of 15) from California and 1 from Venezuela yielded one main RFLP pattern with evidence of two subgroups. The other two isolates (both from patients in the San Joaquin Valley of California) yielded a different RFLP pattern.     

Plasmid analysis as a means of strain differentiation in Clostridium perfringens.

A total of 114 Clostridium perfringens isolates were serotyped and examined for plasmids. Fifty-two strains were from hospitalized patients with diarrhea or from hospital environments, and 62 epidemiologically unrelated isolates were obtained from food poisoning outbreaks. All strains were screened for bacteriocin production against a common indicator strain of C. perfringens. In the one significant hospital outbreak of C. perfringens diarrhea, three to five plasmid types were found in strains of the predominant serotype, but no similar correlation between serotype and plasmid type was found in random isolates from a variety of sources. All of the strains associated with the diarrhea outbreak produced bacteriocins, whereas 63% of the strains from various sources produced bacteriocins. The typing data suggest a promising differentiating capability for plasmid analysis in the epidemiological study of outbreaks of food poisoning, diarrhea, or infections caused by C. perfringens.     

Characterization of Gardnerella vaginalis and G. vaginalis-like organisms from the reproductive tract of the mare.

Gardnerella vaginalis has been isolated from women with bacterial vaginosis, from the genital tracts of asymptomatic women, and from several other infected body sites in humans. However, until recently, it has not been isolated from any other animal species. Between June 1988 and October 1989, 31 isolates identified as G. vaginalis and 70 isolates identified as G. vaginalis-like organisms have been recovered from the genital tracts of 93 mares from Michigan and Ohio. Identification was based on biochemical reactions, hemolysis on media containing blood from various animal sources, and susceptibility to select antimicrobial agents. This report details the characterization of G. vaginalis and G. vaginalis-like organism isolates obtained from the reproductive tracts of these mares and compares the equine isolates with human isolates.     

Characterization of Vibrio cholerae protease activities with peptide digest analysis.

A simple method for the analysis of microbial proteases is described that was used to characterize the proteolytic activities of various Vibrio cholerae isolates. This method utilized the unique peptides generated from the degradation of a standard protein by proteases of various specificities. These peptides were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The unique patterns of peptides seen in gels can be used to type proteases according to their relative specificities. Culture supernatants of V. cholerae isolates from a variety of environmental and human sources were analyzed for the presence of a protease previously isolated and characterized in this laboratory from V. cholerae strain CA401. Supernatants from most isolates showing dimethyl casein proteolytic activity exhibited the presence of enzymes similar to the CA401 protease in their peptide digest patterns against bovine serum albumin and in their immunological reactivities. The probable widespread presence of this virulence-associated protease in V. cholerae isolates is discussed.     

Quinupristin-dalfopristin resistance in Enterococcus faecium isolates from humans, farm animals, and grocery store meat in the United States

Three hundred sixty-one quinupristin-dalfopristin (Q-D)-resistant Enterococcus faecium (QDREF) isolates were isolated from humans, turkeys, chickens, swine, dairy and beef cattle from farms, chicken carcasses, and ground pork from grocery stores in the United States from 1995 to 2003. These isolates were evaluated by pulsed-field gel electrophoresis (PFGE) to determine possible commonality between QDREF isolates from human and animal sources. PCR was performed to detect the streptogramin resistance genes vatD, vatE, and vgbA and the macrolide resistance gene ermB to determine the genetic mechanism of resistance in these isolates. QDREF from humans did not have PFGE patterns similar to those from animal sources. vatE was found in 35%, 26%, and 2% of QDREF isolates from turkeys, chickens, and humans, respectively, and was not found in QDREF isolates from other sources. ermB was commonly found in QDREF isolates from all sources. Known streptogramin resistance genes were absent in the majority of isolates, suggesting the presence of other, as-yet-undetermined, mechanisms of Q-D resistance.     

我国Q热立克次体分离株cbhE‘和cbbE’基因的扩增

用聚合酶链反应（ＰＣＲ）首次对我国Ｑ热立克次体分离株质粒上的特异ＤＮＡ片段ｃｂｈＥ＇和ｃｂｂＥ＇基因进行扩增，目的序列分别为２５８ｂｐ和１４８５ｂｐ。实验结果显示，在我国分离的８株Ｑ热立克次体株中，３株出现了２５８ｂｐ阳性扩增带，５株有１４８５ｂｐ扩增带；两对引物只扩增Ｑ热立克次体的ＤＮＡ。研究证明，我国分离的Ｑ热立克次体株也分别带有ＱｐＨＩ和ＱｐＲＳ型质粒，存在急、慢性分离株之分，但分离株所含质粒类型并不完全与其菌株感染的临床表现相吻合。     

Genetic characterization indicates that a specific subpopulation of Pseudomonas aeruginosa is associated with keratitis infections

Pseudomonas aeruginosa is a common opportunistic bacterial pathogen that causes a variety of infections in humans. Populations of P. aeruginosa are dominated by common clones that can be isolated from diverse clinical and environmental sources. To determine whether specific clones are associated with corneal infection, we used a portable genotyping microarray system to analyze a set of 63 P. aeruginosa isolates from patients with corneal ulcers (keratitis). We then used population analysis to compare the keratitis isolates to a wider collection of P. aeruginosa from various nonocular sources. We identified various markers in a subpopulation of P. aeruginosa associated with keratitis that were in strong disequilibrium with the wider P. aeruginosa population, including oriC, exoU, katN, unmodified flagellin, and the carriage of common genomic islands. The genome sequencing of a keratitis isolate (39016; representing the dominant serotype O11), which was associated with a prolonged clinical healing time, revealed several genomic islands and prophages within the accessory genome. The PCR amplification screening of all 63 keratitis isolates, however, provided little evidence for the shared carriage of specific prophages or genomic islands between serotypes. P. aeruginosa twitching motility, due to type IV pili, is implicated in corneal virulence. We demonstrated that 46% of the O11 keratitis isolates, including 39016, carry a distinctive pilA, encoding the pilin of type IV pili. Thus, the keratitis isolates were associated with specific characteristics, indicating that a subpopulation of P. aeruginosa is adapted to cause corneal infection.     

Vancomycin-resistant gram-positive bacteria isolated from human sources

Recent reports of infections with vancomycin-resistant gram-positive bacteria prompted us to study vancomycin-resistant isolates from human sources to characterize the types of bacteria displaying this phenotype. Thirty-six vancomycin-resistant gram-positive isolates, 14 from clinical specimens and 22 from stool samples, were identified. These isolates were tentatively identified as Lactobacillus spp. (25 strains), Leuconostoc spp. (6 strains), and Pediococcus spp. (3 strains) on the basis of morphology and physiological tests. Two isolates of indeterminate morphology could not be unambiguously assigned to a genus. Four isolates of vancomycin-resistant lactobacilli from normally sterile body sites were considered to be clinically significant. Vancomycin-resistant gram-positive bacteria may represent an emerging class of nosocomial pathogens. Better methods for distinguishing the various genera in the clinical microbiology laboratory are needed.     

Multilocus sequence typing of Candida albicans isolates from animals

Multilocus sequencing strain types of a panel of 43 Candida albicans isolates from animals, including mammals and avian species, were compared with strain types for human isolates. The clade distribution of the animal isolates was significantly different from that of the human isolates, in both a comparison involving a total of 1580 isolates from multiple geographical sources and a comparison restricted to 675 human isolates from the same geographical regions as the animal isolates. A nearest-neighbour analysis involving the 43 animal isolates and 67 human isolates, randomly selected to give a proportionate distribution of geographical sources, showed a strong statistical trend towards genetic selection of different C. albicans strain types adapted to non-human animal hosts, but without complete genetic separation.     

Linkage of the horizontally acquired ypm and pil genes in Yersinia pseudotuberculosis

The superantigen-encoding ypm gene and the pil gene cluster governing type IV pilus biogenesis have been laterally acquired by Yersinia pseudotuberculosis. PCR assays on 270 unrelated strains from various environmental and animal sources revealed a significant association of ypm and pil in isolates.     

Biotyping of Aeromonas isolates as a correlate to delineating a species-associated disease spectrum.

A group of 147 Aeromonas isolates from diverse clinical and environmental sources was subjected to the biotyping scheme of Popoff and Veron. Of the 147 isolates biotyped, 137 (93%) could be identified, with Aeromonas hydrophila predominating (48%) and equal percentages (25 to 27%) of the other two species (Aeromonas sobria and Aeromonas caviae). A number of additional biochemical properties were found to be significantly associated with one or more of these three species. These included lysine decarboxylase activity, hemolysis of sheep erythrocytes, lecithinase production, staphylolytic activity, arbutin hydrolysis, and acid production from utilization of various carbohydrates. By incorporating these phenotypic properties into an extended biotyping system, 98% of the isolates were identified. Selective distribution of individual species with respect to certain body sites was noted.     

Direct hemagglutination technique for differentiating Bacteroides asaccharolyticus oral strains from nonoral strains.

A simple and economical method for differentiating Bacteroides asaccharolyticus of oral sources from nonoral sources is described. The present data indicate that oral strains of B. asaccharolyticus strongly agglutinate sheep erythrocytes, whereas isolates from various nonoral sites typically are devoid of hemagglutination activity. The direct hemagglutination test may aid in determining the source of B. asaccharolyticus present in an infection, and thus the procedure has potential value as a means of biotyping.     

Biofilm formation in clinical isolates of group B streptococci from north India

Many bacterial species are capable of living as biofilm, thought to be the predominant growth mode for bacteria in natural environments. Increasing evidence implicates biofilm as the cause of various human infections. In this study, biofilm formation was demonstrated in group B streptococci (GBS) isolated from different sources in the north Indian community at various pH ranges as well as sugar concentrations and correlated it with different serotypes and surface gene (c alpha) profiles. The capability to form biofilm was better demonstrated in strains from asymptomatic carriers (pregnant women) compared to symptomatic patients. Quantitatively bacterial adherence with host cells was greater in isolates that produced biofilm under neutral conditions. This study assessed the biofilm formation in clinical isolates of GBS, which is a step towards understanding its role in pathogenesis.     

Biotyping of Serratia marcescens and its use in epidemiological studies.

A Serratia marcescens biotyping system using eight carbon sources (benzoate, DL-carnitine, m-erythritol, 3-hydroxybenzoate, 4-hydroxybenzoate, lactose, D-quinate, and trigonelline), a tetrathionate reduction test, production of prodigiosin, and horse blood hemolysis was derived from a recent numerical taxonomic study (Grimont et al., J. Gen. Microbiol. 98:39-66, 1977). A total of 98.6% of 2,210 isolates from various sources could be assigned to 1 of 19 biotypes. Distribution and spread of 1,088 S. marcescens isolates throughout 13 clinical departments of Pellegrin Hospital (Bordeaux, France) were studied from 1968 through 1975. Except for one that colonized the intestinal tract of newborns, the six pigmented biotypes were seldom isolated. Each of the 13 nonpigmented biotypes showed a particular pattern of distribution and spread. The usefulness of S. marcescens biotyping was shown by relating several isolates recovered from patients and their inanimate environment and by pointing out the possible existence of infections or colonizations by two unrelated biotypes. S. marcescens strains isolated from the natural environment (water) are usually pigmented, and their biotypes are uncommon in hospitals. Biotyping can, therefore, be of help in epidemiological and ecological surveys.     

Identification of heterogeneity in human isolates of Giardia lamblia by isoenzyme studies.

Electrophoretic mobility patterns of six enzymes, viz. alkaline phosphatase E.C. 3.1.3.1., acid phosphatase E.C. 3.1.3.2., malic enzyme E.C. 1.1.1.40., phosphoglucomutase E.C. 2.7.5.1., isocitrate dehydrogenase E.C. 1.1.1.42., glucose-6-phosphate dehydrogenase E.C. 1.1.1.49 of two axenically cultured human Giardia lamblia isolated from India (PD-1 and PD-2) and one strain from Portland, Oregon, USA (P-1) were compared using polyacrylamide gel electrophoresis (PAGE). Based on the difference in the mobility patterns of the enzymes phosphoglucomutase, isocitrate dehydrogenase and malic enzyme, the PD-1 and PD-2 isolates appeared to be quite different from P-1. In the present study, the isocitrate dehydrogenase and alkaline phosphatase enzymes were used for the first time for differentiation of Giardia isolates. In the case of PD-1, two alkaline phosphatase bands could be seen whereas only one band was observed in PD-2 and P-1. Thus, the three strains could be grouped into three different zymodemes. These findings reveal the significant heterogeneity in G. lamblia isolates both from widely separated areas and within a single region. Heterogeneity among G. lamblia strains may explain the variable clinical manifestations, host response and treatment efficacy characteristic of human giardiasis.     

Pseudomonas aeruginosa enzyme profiling: predictor of potential invasiveness and use as an epidemiological tool

The enzymatic potential of 54 clinical and 22 environmental isolates of Pseudomonas aeruginosa from soil and water were evaluated by substrate plate assays. Clinical isolates produced substantial levels of 9 of the 11 enzymes assayed, whereas strains recovered from soil or water were relatively inert enzymatically. Elastase, deoxyribonuclease, and elevated protease activities were associated preferentially with clinical isolates of systemic origin; these activities were found twice as frequently in clinical isolates as in strains derived from sputum or the urogenital tract. Our data suggest that these factors may play an important role in the dissemination of P. aeruginosa from local or superficial sites. A comparison of the enzyme profiles of the environmental and clinical isolates indicated that colonization or infection by environmental strains of P. aeruginosa is a rare event and that environmental and clinical strains comprise separate biovars. Epidemiologically, enzyme profiles permitted the fingerprinting and differentiation of clinical strains from various sources.