Multilocus Enzyme Genotypes Research Articles

Clonal Population Structure of Colombian Sylvatic Trypanosoma cruzi

Isoenzyme variability and evidence of genetic exchange were evaluated in 75 wild stocks of Trypanosoma cruzi obtained from different hosts from 5 geographical regions within the endemic area in Colombia. Cluster analysis of genetic variability was attempted. Thirty-three multilocus enzyme genotypes (clonets) were identified from 75 stocks, 27 of which clustered with zymodeme Z1 and 6 with zymodeme Z3. Two stocks isolated from human infections showed the potential risk to rural communities in Colombia. The stocks exhibited departures from Hardy-Weinberg expectations, including both fixed heterozygote and fixed homozygote demes, where both segregation and recombination were absent. To inspect for population subdivision that might falsely imply clonality in these stocks, Wright's F statistics were calculated. Theta values (Fst) were significantly different from 0 when 33 clonets, 27 Z1-like clonets, and 5 geographical subpopulations were compared; thus, a significant amount of divergence has occurred between and within them. In addition, linkage disequilibrium was detected for most possible pairwise comparisons of loci. In conclusion, the above results all support a scenario of long-term clonal evolution in Colombian sylvatic T. cruzi populations.

Subtyping of common pediatric pneumococcal serotypes from invasive disease and pharyngeal carriage in Finland.

Ninety-nine penicillin-sensitive Streptococcus pneumoniae strains of common pediatric serogroups/types (6, 7, 14, 19, and 23) cultured from the blood of children with invasive disease (n = 49) or asymptomatic oropharyngeal carriage (n = 50) were analyzed by multilocus enzyme electrophoresis and ribotyping; 53 distinctive multilocus enzyme genotypes (ETs) and 53 ribotypes were identified. Multilocus enzyme electrophoresis showed good correlation with ribotying. ETs and ribotypes among invasive and carriage isolates were similar. Within different S. pneumoniae serogroups/types, both clonal (7 and 14) and heterogenous (6, 19, and 23) ET and rRNA hybridization patterns were observed. Greatest diversity was observed among serotypes 6A, 6B, and 19F. Use of 12 additional restriction enzymes besides PvuII and BglII did not increase ribotype discrimination within serotype 7 and 14 isolates. Serotype 7 and 14 strains, which appear clonal by subtyping, are rare among carriers but common causes of invasive disease. Characteristics associated with their clonality may represent an advantage for invasiveness.

Characterization of Streptococcus agalactiae strains by multilocus enzyme genotype and serotype: identification of multiple virulent clone families that cause invasive neonatal disease

The chromosomal genotypes of 277 isolates of 16 serotypes of Streptococcus agalactiae were characterized by analysis of electrophoretically demonstrable allele profiles at 12 metabolic enzyme loci. The collection comprised the type strain and 276 strains recovered from French symptomatic and asymptomatic subjects. Sixty-one distinctive electrophoretic types (ETs), representing multilocus clonal genotypes, were identified. Cluster analysis of the ETs revealed two primary phylogenetic divisions separated by a genetic distance of 0.62, Division I contained 67 isolates which could be assigned to 13 ETs. Twenty-seven of these isolates were from samples of cerebrospinal fluid (CSF) from neonatal meningitis patients. Two ETs, separated by a genetic distance of 0.217, contained 26 of these 27 isolates. Division II contained 210 isolates, of which 27 were isolated from CSF. This division was more polymorphic and included 48 ETs. Spanning a genetic distance of 0.3, three clusters and one ET were identified within this group. Twenty-four of 27 strains isolated from CSF belonged to one cluster, and 19 of them belonged to two adjacent ETs with a genetic distance of 0.083. Fifty-five of the 68 serotype Ia strains and 24 of the 26 serotype Ib strains were each confined to one of the evolutionary lineages, and 85 of the 86 strains which carried protein antigen c belonged to phylogenetic division II. Most of the type III organisms were assigned to two clone families. The characteristics of this French population argue for the existence of particular groups of strains responsible for neonatal meningitis and demonstrate that serotyping can supply information about the genetic distribution of strains.

Nosocomial transmission of disease caused by nontypeable strains of haemophilus influenzae

purpose: The authors evaluated a geographic and temporal cluster of lower respiratory tract infections due to unencapsulated (serologically nontypeable) Haemophilus influenzae to determine whether this event represented the transmission of a single clone. methods and materials: H influenzae was recovered from eight patients at a nursing home and from three patients in an adjacent acute care hospital. Serotypes, biotypes, outer membrane protein profiles, and multilocus enzyme genotypes were determined to characterize bacterial isolates. Patient records were retrospectively examined to determine clinical and epidemiologic characteristics. results: During a 10-day period in September 1991, lower respiratory tract infections caused by H influenzae were diagnosed in four patients residing in a single nursing home unit. Oropharyngeal cultures from four of seven asymptomatic roommates of these patients also grew H influenzae. During the month before and after the nursing home cluster of cases, four other individuals in acute care areas of the hospital had positive sputum cultures for H influenzae. Three of these latter specimens were also available for analysis. All H influenzae isolates were unencapsulated and β-lactamase-negative. Eight of the nine isolates from the nursing home patients (two morphologically distinct colony types of H influenzae were isolated from one case) had a single outer membrane protein profile arbitrarily designated as X and a single multilocus enzyme genotype arbitrarily designated as A. In contrast, none of the isolates from the acute care cases had this profile ( P <-0.02; two-tailed Fisher's exact test). The isolates obtained from two of the patients in acute care areas had an outer membrane protein profile arbitrarily designated as Y and a single multilocus enzyme genotype designated as B. These two patients were contemporaneously hospitalized in adjacent intensive care unit cubicles. The remaining isolates displayed an outer membrane protein profile arbitrarily designated as W. All roommates of the four patients in the nursing home were administered oral rifampin 600 mg daily for 4 days. H influenzae was not recovered from follow-up oropharyngeal cultures obtained 1 week after the completion of therapy. No β-lactamase-negative H influenzae were identified in this unit during the subsequent 9 months. conclusion: This study furnishes strong evidence for the nosocomial transmission of a clone of unencapsulated H influenzae in a nursing home unit. Epidemiologic data showed temporal and geographic clustering of respiratory tract infections and colonization by H influenzae. Outer membrane protein profiles and multilocus enzyme genotype analysis indicated that seven of eight patients at the nursing home carried a single clone of unencapsulated H influenzae. Laboratory and epidemiologic data also demonstrated the presence, and possible nosocomial transmission, of a second clone of unencapsulated H influenzae in a physically separate area of the hospital. Finally, although a causal relationship is not proven, the outbreak ended following the administration of rifampin prophylaxis of asymptomatic carriers.

Evidence for the introduction of a multiresistant clone of serotype 6B Streptococcus pneumoniae from Spain to Iceland in the late 1980s.

Almost all of the multiresistant pneumococci that appeared suddenly in clinical specimens in Iceland between 1989 and 1992 belonged to serogroup 6. Fifty-seven of these isolates were analyzed for serotype, penicillin-binding protein pattern, multilocus enzyme genotype, and fragmentation pattern obtained by pulsed-field electrophoretic separation of restriction enzyme digests of chromosomal DNA. All isolates were of serotype 6B and had similar or identical patterns in each molecular test. The Icelandic isolates were indistinguishable from a subgroup of multiresistant serotype 6B pneumococci that has been present with high incidence in Spain during the past two decades. The data suggest the import to Iceland of a single multiresistant clone of pneumococcus, most likely from Spain.

Evolutionary origin and radiation of the avian-adapted non-motile salmonellae

Multilocus enzyme electrophoresis was employed to estimate chromosomal genotypic diversity and relationships among 131 isolates of the non-motile Salmonella biotypes Gallinarum and Pullorum (serotype 1, 9, 12:-:-) that cause fowl typhoid and pullorum disease, respectively. Thirteen electrophoretic types (ETs), marking clones, were distinguished, and construction of a neighbour-joining phylogenetic tree revealed three lineages: one consisted of five ETs of Gallinarum, a second included seven ETs of Pullorum, and a third was represented by a single ET (Ga/Pu 1) that is intermediate between those of the other two lineages in both multilocus enzyme genotype and biochemical properties. Enzyme genotype analysis and comparative nucleotide sequencing of the phase 1 flagellin gene (fliC), the hook-associated protein 1 gene (flgK), and the 6-phosphogluconate dehydrogenase gene (gnd) identified serotype Enteritidis (1, 9, 12:g, m:-) as a close relative of the non-motile salmonellae. In most strains of biotype Gallinarum, the fliC gene is complete, intact and identical in sequence to that of Enteritidis, but isolates of three ETs had a stop codon at position 495. The fliC sequences of the ETs of Pullorum differed from that of Enteritidis in having non-synonymous changes in either two or three codons and a synonymous change in one codon. The sharing of distinctive alleles at three metabolic enzyme loci and a stop codon in flgK indicates that the non-motile salmonellae are monophyletic and that their most recent common ancestor was non-motile. Since diverging from that ancestor, the Pullorum lineage has evolved more rapidly than the Gallinarum and Ga/Pu 1 lineages.

Geographic and temporal distribution and molecular characterization of two highly pathogenic clones of Streptococcus pyogenes expressing allelic variants of pyrogenic exotoxin A (Scarlet fever toxin).

The molecular population genetics and pathogenic potential of North American and European invasive strains of Streptococcus pyogenes were assessed. Isolates from recent invasive infections and from infections in the 1920s and 1930s were characterized for multilocus enzyme genotype and allelic variation in the gene (speA) that encodes streptococcal pyrogenic exotoxin (SPE) A (scarlet fever toxin). A subset of strains was studied for allelic variation in genes that encode SPE B and streptokinase. All contemporary strains assigned to electrophoretic types (ETs) 1 and 2 that synthesize SPE A have the speA2 and speA3 allelic variants, respectively, and their relative virulence in two mouse models is similar to that of strains of the same ET and M protein types recovered earlier. In contrast, ET 1 and 2 isolates from disease episodes in the 1920s and 1930s contain the speA1 allele. The data suggest there may be temporal and geographic variation in the occurrence of clone--virulence factor allele combinations, an observation that may in part explain fluctuations in disease frequency, severity, and character.

Clonal analysis of methicillin-resistant Staphylococcus aureus strains from intercontinental sources: association of the mec gene with divergent phylogenetic lineages implies dissemination by horizontal transfer and recombination.

Genetic relationships among 254 isolates of Staphylococcus aureus resistant to methicillin recovered between 1961 and 1992 from nine countries on four continents were determined by analyzing electrophoretically demonstrable allelic variation at 15 chromosomal enzyme loci. Fifteen distinctive electrophoretic types, marking clones, were identified. The mec gene is harbored by many divergent phylogenetic lineages representing a large portion of the breadth of chromosomal diversity in the species, a result that is interpreted as evidence that multiple episodes of horizontal transfer and recombination have contributed to the spread of this resistance determinant in natural populations. Isolates recovered in the United Kingdom, Denmark, Switzerland, Egypt, and Uganda in the 1960s are of a single multilocus enzyme genotype and probably are progeny of an ancestral methicillin-resistant clone. There is geographic variation in the frequency of recovery of the common methicillin-resistant clones, an observation that may in part explain reported regional differences in natural history correlates of resistant organisms.

Geographic distribution of penicillin-resistant clones of Streptococcus pneumoniae: characterization by penicillin-binding protein profile, surface protein A typing, and multilocus enzyme analysis.

Examination of several hundred penicillin-resistant clinical isolates of Streptococcus pneumoniae has revealed extensive strain-to-strain variation in the number and molecular size of penicillin-binding proteins (PBPs). This polymorphism has been used to classify resistant isolates into groups (PBP families) that share distinct electrophoretic profiles. We describe herein properties of four such PBP families: two from Spain (and/or Ohio) and one each from Hungary and Alaska. We have discovered that representative isolates assigned to each PBP family also share capsular serotype, antibiotic resistance pattern, pneumococcal surface protein A type, and multilocus enzyme genotype. The results demonstrate independent clonal origin for strains assigned to each PBP family. Each resistant clone occurs with uniquely high incidence within specific geographic areas.

Streptococcus pyogenes Pharyngitis: Characterization of Strains by Multilocus Enzyme Genotype, M and T Protein Serotype, and Pyrogenic Exotoxin Gene Probing

<i>Streptococcus pyogenes</i> Pharyngitis: Characterization of Strains by Multilocus Enzyme Genotype, M and T Protein Serotype, and Pyrogenic Exotoxin Gene Probing

Emergence of a new clone of serogroup C Neisseria meningitidis in São Paulo, Brazil.

Serogroup C isolates of Neisseria meningitidis recovered from 121 patients with meningitis or septicemia in Greater São Paulo, Brazil, between 1976 and 1990 were analyzed with respect to serotype and multilocus enzyme genotype. The distribution of serotypes has changed since 1989 when serotype 2b started to replace serotype 2a. There were 48 distinct multilocus genotypes (electrophoretic types [ETs]) and 13 distinct complexes. Among the 41 serotype C:2b:- strains analyzed, 38 (93%) were found in complex 11. The percentage of complex 11 increased from 8% in 1988 to 50 and 66% in 1989 and 1990, respectively. Although we have been in an epidemic situation due to serogroup B:4:P1.15 ET-5 complex since 1988, the appearance and increase of a new unrelated strain, C:2b:- of ET-11 complex, in 1989 and 1990 provide enough data to conclude that the presence of two different complexes, ET-5 and -11, of N. meningitidis were responsible for the high levels of meningococcal disease in Greater São Paulo during this period.

Evidence consistent with horizontal transfer of the gene (emm12) encoding serotype M12 protein between group A and group G pathogenic streptococci.

Human isolates of Lancefield group G streptococci harbor sequences homologous with the structural gene (emm) encoding M protein, a major virulence factor in Streptococcus pyogenes (a group A Streptococcus species). We used DNA-DNA hybridization, restriction endonuclease chromosomal profiling, and multilocus enzyme electrophoresis to examine genetic relationships between group A and group G streptococcal strains expressing homologous serologic type 12 M (M12) protein. All M12 group A strains studied had very similar restriction endonuclease genomic profiles and multilocus enzyme genotypes. In contrast, the restriction enzyme genomic profile and multilocus enzyme genotype of the M12 group G strain CS140 were strikingly different from those characterizing the M12 group A organisms. DNA-DNA hybridization studies revealed, on average, 57% genomic similarity between the M12 group A and group G strains. Taken together, our data demonstrate that a gene encoding M12 protein occurs in two highly divergent chromosomal backgrounds, a result suggesting that an episode of horizontal gene transfer and recombination has occurred between two streptococcal lineages.

Molecular polymorphism and epidemiology of Neisseria meningitidis immunoglobulin A1 proteases.

Neisseria meningitidis is one of several important bacterial pathogens that secrete a specific protease capable of cleaving human immunoglobulin A1 (IgA1) in the hinge region. To obtain further information on this putative virulence factor, we examined the IgA1 protease and iga gene region of 133 isolates of N. meningitidis assigned to 88 multilocus enzyme genotypes and representing major epidemics and carrier strains from 19 countries. Of the two IgA1 cleavage specificities previously observed, isolates associated with epidemics of meningococcal disease showed exclusively type 1 IgA1 protease activity. Considerable heterogeneity of the N. meningitidis IgA1 protease was demonstrated at both the protein and gene levels. Thus, five different forms of IgA1 protease were detected with enzyme-neutralizing antibodies raised in rabbits. An antiserum raised against a single type 2 IgA1 protease inhibited the enzyme activity of all strains examined, a finding of potential significance for the possible application of IgA1 protease in a vaccine against meningococcal disease. Examination of the iga gene region with restriction endonucleases revealed a high degree of polymorphism among strains belonging to some multilocus enzyme genotypes. The different iga gene types did not correlate with cleavage type or inhibition of the IgA1 protease. Our findings indicate that horizontal genetic exchange occurs in vivo with considerably different frequency in different clones of meningococci.

Streptococcus pyogenes pharyngitis: characterization of strains by multilocus enzyme genotype, M and T protein serotype, and pyrogenic exotoxin gene probing.

Multilocus enzyme electrophoresis, serological characterization of M and T proteins, and probing for pyrogenic exotoxin A and C genes were used to investigate the bacteriologic epidemiology of strains of Streptococcus pyogenes recovered primarily from patients with recurrent pharyngitis. A total of 164 strains recovered from individuals living in nine states of the United States was analyzed. Two-thirds of the patients in our sample were infected with the homologous strain following antibiotic therapy and presumably represented treatment failures, whereas the other one-third of the patients were infected with a heterologous strain after therapy and probably represented reinfections. Multilocus enzyme electrophoresis was as efficacious in strain discrimination as serologic typing techniques were and, in addition, successfully characterized all organisms that were serologically nontypeable. Two clones of S. pyogenes responsible for most of the episodes of toxic shock-like syndrome in the United States are geographically widespread, but they vary by locality in the frequency of their occurrence. Compared with a sample of strains cultured from patients whose pharyngeal infections were eliminated by antimicrobial therapy, these two clones were statistically overrepresented among organisms that cause recurrent pharyngitis.

Characterization of serogroup A and B strains of Neisseria meningitidis with serotype 4 and 21 monoclonal antibodies and by multilocus enzyme electrophoresis

The reactions of serogroup A strains of Neisseria meningitidis with one monoclonal antibody specific for serotype 21 and three different monoclonal antibodies specific for serotype 4 were compared with those of serogroup B strains previously assigned to serotype 4. Antibody binding was studied by enzyme-linked immunosorbent assay (ELISA), dot blotting, and immunoblotting. Characterization of the isolates by the electrophoretic mobilities of 14 metabolic enzymes showed 50 multilocus enzyme genotypes. All except two genotypes fell into three distinct clusters: I, IIa and IIb. The enzyme genotypes of serogroup B strains were mainly in cluster I, and 88% of the serogroup A strains had genotypes in clusters IIa and IIb. Serogroup B strains generally reacted with all three serotype 4 monoclonal antibodies in ELISA and dot blotting but with only two in immunoblots. Serogroup A strains showed two different reactions in the blotting methods: either binding of the serotype 21 antibody only or binding of this and two of the three serotype 4 monoclonal antibodies. Strains of the first pattern were in clusters I and IIa, whereas all but two strains in cluster IIb were of the second pattern. In ELISA, an additional reaction of two of the serotype 4 monoclonal antibodies with serogroup A isolates was observed. The different binding of these two monoclonal antibodies in ELISA and the blotting methods appeared to result from heat inactivation of the meningococcal cells and use of detergent-containing reagents in ELISA. The results show that the serotype of serogroup A strains is distinct from serotype 4 of serogroup B strains.

Characterization ofNeisseria meningitidis serogroup A strains from an outbreak in France by serotype, serosubtype, multilocus enzyme genotype and outer membrane protein pattern

In an attempt to determine the epidemiological relationship between cases of infection caused by Neisseria meningitidis serogroup A in France between August 1987 and December 1988, and an outbreak which occurred in Mecca in August 1987, markers such as serotype, serosubtype, multilocus enzyme genotype and outer membrane protein (OMP) pattern were used to characterize the bacterial isolates. From a total of 28 cases, 20 strains were isolated. Sixteen isolates, including one which undoubtedly originated from the Mecca epidemic, were homogenous (serotype 4, serosubtype P1.9, multilocus enzyme genotype of clone III-1, identical OMP pattern). Four isolates had a different OMP pattern with little or no P1 protein, no P4 and no P6. Two of these isolates belonged to other clones (I.1 and group I) and they were considered to be unrelated to the Mecca epidemic strains. The origin of the remaining two isolates was not clear. It is concluded that the majority of the Neisseria meningitidis serogroup A strains recently isolated in France originate from the Mecca epidemic.

Clones of Serogroup B Neisseria meningitidis Causing Systemic Disease in the Netherlands, 1958-1986

Serogroup B isolates of Neisseria meningitidis recovered from 278 patients with systemic disease in the Netherlands between 1958 and 1986 were analyzed with respect to serotype and multilocus enzyme genotype. Of the isolates, 28% were serotype 2b and 53% were neither serotypeable nor serosubtypeable. There were 145 distinct multilocus genotypes (electrophoretic types, ETs), with up to 31 isolates belonging to the same ET. Temporal changes in the genotypic composition of meningococcal populations in the Netherlands were demonstrated by the recent occurrence of disease caused by three clone lineages, I, III, and VI, that were not found before 1975. The epidemic of 1966-1967 and the hyperendemic wave of 1972 were caused, in large part, by two closely related but distinct clones of serotype 2b isolates, ET-11 and ET-17, respectively. Deviations in male-to-female ratio and age distribution of patients were observed for disease caused by isolates of individual clone lineages.

Frequent Acquisition of Multiple Strains of Methicillin-Resistant Staphylococcus aureus by Healthcare Workers in an Endemic Hospital Environment

Methicillin-resistant Staphylococcus aureus (MRSA) has been an endemic nosocomial pathogen at the VA medical center (VAMC) in Providence, Rhode Island since 1981. From 1985 to 1987, more than 30% of all unique S aureus isolates were methicillin resistant. To evaluate the frequency of acquisition of MRSA isolates by healthcare workers, we compared the antimicrobial susceptibility patterns, multilocus enzyme genotypes and plasmid profiles of isolates recovered from nasal and hand cultures from VAMC nurses and house staff on rotation at the VAMC with those of clinical isolates from patients at the VAMC and four other affiliated hospitals. Fifty-six percent of ward nurses cultured (n = 112) were colonized with S aureus, of which 65% was methicillin resistant. Six isolates of MRSA were identified on the initial culturing of house staff (n = 65); 16 MRSA isolates were recovered at the end of a four-week rotation (p less than .02). Phenotypic and genotypic analyses demonstrated that numerous distinct MRSA strains were recovered in the study period. The incidence of MRSA among clinical isolates at the VAMC and affiliated institutions was remarkably constant throughout the three-year study period. Moreover, despite regularly sharing resident physicians, interns and medical students, MRSA isolates were commonly recovered at the other university-affiliated hospitals. Our study failed to reveal evidence of significant interhospital transmission of MRSA isolates by healthcare workers. While healthcare workers may contribute to the dissemination of MRSA within institutions, they appear to be less important in spreading MRSA between institutions.

Genotypic heterogeneity of strains of Citrobacter diversus expressing a 32-kilodalton outer membrane protein associated with neonatal meningitis

Genetic diversity and relationships among 42 strains of Citrobacter diversus recovered from the cerebrospinal fluid of human infants with meningitis and from other clinical sources in the United States were estimated on the basis of electrophoretically detectable allelic variation in 20 genes encoding metabolic enzymes. Sixteen distinctive multilocus enzyme genotypes were identified, among which the mean genetic diversity per locus was 0.244. The recovery of isolates of the same genotype in several regions of the United States and over periods as long as 20 years indicates that the population structure of C. diversus is clonal. There was little association between multilocus enzyme genotype and biotype, piliation, or presence of a 32-kilodalton outer membrane protein. The observation that the 32-kilodalton outer membrane protein, which is expressed predominantly by strains recovered from infants with meningitis, occurs in a variety of genotypically diverse clones belonging to several phylogenetic lineages supports the hypothesis that this protein confers virulence.

Characterization of epidemic and nonepidemic Neisseria meningitidis serogroup A strains from Sudan and Sweden

A random selection of 25 strains isolated during an epidemic caused by serogroup A Neisseria meningitidis in Sudan (1988), 3 preepidemic meningococcal strains (1985), and 26 serogroup A strains isolated from sporadic cases of meningitis in Sweden (1973 to 1987) were assessed for multilocus enzyme genotypes (ETs), DNA restriction enzyme patterns, outer membrane proteins, lipopolysaccharides, pilus formation, and antibiograms. All of the 25 Sudanese epidemic isolates and 22 of the Swedish strains were of the same or closely related ETs (ETs 3, 4, and 5), corresponding to clone III-1, which has been responsible for two pandemic waves in the last three decades. The earlier pandemic involved Scandinavia, and the last one caused an outbreak during the pilgrimage to Mecca, Saudi Arabia (August 1987), spreading to Sudan, Chad, and Ethiopia. The three Sudanese preepidemic isolates (1985) were clone IV-1 (sulfonamide susceptible), which has been resident in the African meningitis belt for the last 25 years. The uniformity of clone III-1 strains (all sulfonamide resistant) from Sudan and Sweden was confirmed by DNA restriction enzyme patterns. ETs 3, 4, and 5 from Sudan and Sweden had 86 to 100% similarity to a Swedish clone III-1 reference strain, whereas ETs 1, 2, 6, and 7 showed 50 to 80% similarity. Class 1 protein for clone III-1 showed serosubtype antigens P1.9 and P1.x, whereas ET6 strains (clone IV-1) had serosubtype P1.7. Lipopolysaccharides were variable in the Sudanese and Swedish strains. Pili were expressed in all clone III-1 isolates from Sudan and Sweden but in none of the clone IV-1 isolates (Sudan, 1985).