Epidemic Isolates Research Articles

Codominant Mixtures of Viruses in Reference Strains of Influenza Virus Due to Host Cell Variation

Influenza viruses grown in chicken eggs may comprise mixtures of variants, creating problems in establishing international reference strains and in preparing high growth reassortants. We therefore analyzed representative reference strains of H3N2 viruses from 1987 to 1989 by direct sequencing of HA1. Three of seven reference strains had different nucleotides at the same position in nucleotide gels, indicating the presence of codominant mixtures. These nucleotide duplications occurred at residues previously shown to code for amino acids associated with egg adaptation (156, 186, and 193 of HA1). Cloning of these viruses in chicken eggs permitted separation of the mixtures, and the majority of these cloned viruses could be distinguished with monoclonal antibodies. The remaining four reference strains were homogeneous and contained one of the two amino acids usually found at these residues in HA1 (e.g., 145 Glu or Lye, 186 Ser or Ile). Analysis of epidemic H3N2 isolates, for which mammalian cell and egg isolates are available for sequence analysis from the same patient, confirmed that multiple nucleotide changes can occur at residues associated with egg adaptation. When reference or epidemic strains containing codominant mixtures were passaged in eggs one to five times, one of the codominant strains usually became dominant. Similar results were obtained with Madin Darby Canine kidney (MDCK) cells, although the dominant virus usually differed from that in eggs. Attempts to reselect an influenza virus possessing sequence changes in HA1 characteristic of mammalian cells or the original human isolate (i.e., 156 Glu, 158 Glu, 186 Ser) by multiple passages in MUCK cells were not successful, but evidence was obtained that MDCK cells can provide a selective growth advantage. Thus, variants that are dominant in eggs are not necessarily dominant in MDCK cells. To preserve the original genotype of viruses used as reference strains, we recommend the following procedure: (i) cloning in chicken eggs of the candidate virus at a very early passage, (ii) selection and analysis of multiple clones with the same ferret polyclonal and/or monoclonal antibodies used in the initial screening, and (iii) selection of the isolate whose hemagglutinin molecule most closely resembles the clinical isolate.

An Introduction to Molecular Hospital Epidemiology

One of the primary goals of the hospital epidemiologist is the identification and characterization of nosocomial outbreaks. Outbreaks usually are identified by the recovery of a unique strain from a cluster of patients infected with a nosocomially acquired pathogen. Until recently, the microbiologic tools available to any hospital epidemiologist permitted identification of novel strains by speciation and antibiogram. Thus, most outbreak descriptions consisted of the identification of an unusual species or the appearance of a new antibiotic resistance phenotype in a recognized nosocomial pathogen. The emergence of enterococci in this decade or the emergence of methicillin-resistant Staphylococcus aureus (MRSA) in the 1970s am notable examples. Where resources have existed, additional methods have been used for further discrimination. Outbreak and endemic strains have been compared by phage typing, serologic typing, and capsular typing. For example, beta-hemolytic streptococci may be grouped by Lancefield antisera, and Staphylococcus aureus may be grouped by phage typing. However, certain Lancefield groups and certain phage groups are common among epidemic and endemic isolates, which may prevent identification of a specific epidemic strain.

Large restriction fragment patterns of genomic Mycobacterium fortuitum DNA as strain-specific markers and their use in epidemiologic investigation of four nosocomial outbreaks.

Pulsed-field gel electrophoresis and restriction endonucleases with rare recognition sites were used to generate large restriction fragment (LRF) patterns of genomic DNA from 48 isolates of Mycobacterium fortuitum biovariant fortuitum. Epidemiologically unrelated isolates gave highly diverse patterns when AsnI, HpaI, AflII, DraI, NdeI, XbaI, SpeI, or SspI was used. Epidemiologically related isolates produced identical or minimally different LRF patterns. Minor variations in LRF patterns were seen in two epidemic isolates digested with XbaI, suggesting that genetic alteration had occurred. LRF patterns were used to study three cardiac surgery wound infection outbreaks and one respiratory disease nosocomial outbreak. In two outbreaks, LRF patterns confirmed the reported clustering of isolates on the basis of multiple phenotyping methods. In the remaining two outbreaks, isolates which could not be separated by prior typing methods were easily distinguished by LRF pattern analysis. Environmental water isolates from two outbreaks had LRF patterns identical to those of the disease-producing strains, confirming that the local environment was the source of infection. Pulsed-field gel electrophoresis of LRFs of genomic DNA offers great promise as an epidemiologic tool for the study of M. fortuitum.

Phylogenetic analysis of a coxsackievirus A24 variant: The most recent worldwide pandemic was caused by progenies of a virus prevalent around 1981

Nucleotide substitutions in the viral-encoded proteinase 3C pro) region (549 nucleotides) of the RNA genome of a coxsackievirus A24 variant (CA24v), one of the agents causing acute hemorrhagic conjunctivitis (AHC), were studied using 32 isolates collected from the Eastern hemisphere in 1970–1989. Based on regression analysis of nucleotide differences among isolates, the nucleotide substitution rate of CA24v 3C pro was estimated to be 3.7 × 10 −3 nucleotide/year. A phylogenetic tree constructed by the modified unweighted pair group method using arithmetic averages (UPGMA) indicated that CA24v had evolved from a common ancestor which appeared in one focal place in November 1963 ± 21 months, about 7 years before the first isolation of CA24v in Singapore. The tree also revealed that all the recent epidemic isolates in 1985–1989 including Asian and Ghanian strains diverged from each other after 1981. This finding is consistent with the evidence that AHC due to CA24v had been confined to Southeast Asia and the Indian subcontinent until 1985, then suddenly and explosively spread to other areas where no CA24v isolations had been reported.

Molecular typing of Salmonella enterica subsp. enterica serovar Wien by rRNA gene restriction patterns

Analysis of digested DNA from 40 Salmonella enterica subsp. enterica serovar Wien isolates revealed five different rRNA gene restriction patterns for HindIII (H1 to H5) and five for PstI (P1 to P5). The isolates had been selected on the basis of the year of isolation, the geographic origin and the antibiotic resistance pattern and were distinguished as follows: a) 13 pre-epidemic isolates from different French towns in 1958–1969 and from Senegal in 1968; b) 7 epidemic isolates from Algiers in 1969; c) 20 post-epidemic isolates from different French and Italian towns in 1970–1990. Three different rRNA patterns (H1P1, P3P3 and H4P4) were observed among the pre-epidemic isolates. Conversely, the epidemic isolates, which were characterized by the previously described large antibiotic resistance patterns and by the presence of 1.3 and 80–109 MDa plasmids, belonged to the same H1P1 ribotype. All but two post-epidemic isolates were of the H1P1 ribotype. The determination of rRNA gene restriction patterns together with the plasmid content proved to be useful for a better characterization of the serovar Wien endemic and epidemic isolates.

Usefulness of three probes in typing isolates of methicillin-resistant Staphylococcus aureus (MRSA)

Fifty-nine epidemiologically unrelated methicillin-resistant Staphylococcus aureus (MRSA) isolates from different geographical areas and 23 phage-type 77 MRSA isolates from France were investigated. Cellular DNA, digested with restriction endonucleases EcoRI or HindIII, was probed with plasmids carrying the gene encoding 16S rRNA (pBA2), the gene aacA-aphD (pSF815A) and the gene aacA-aphD plus part of IS256 (pIP1307). When probed with pBA2, most of the unrelated isolates displayed the same hybridisation pattern. A greater diversity in patterns was detected in gentamicin-resistant strains with the two other probes. The most accurate fingerprinting of these isolates was obtained with the probe pIP1307. Moreover, this probe appeared to be useful for tracing the phage-type 77 epidemic MRSA isolates widespread in French hospitals.

Typing of methicillin resistant and susceptibleStaphylococcus aureus strains by ribosomal RNA gene restriction patterns using a biotinylated probe

Nine methicillin-sensitive (MSSA) and 37 methicillin-resistant (MRSA) Staphylococcus aureus isolates of various phage types and resistotypes from seven countries were investigated. Chromosomal DNA was restricted with HindIII or EcoRI, Southern blotted and hybridised with a cDNA probe to 16S+23S rRNA derived from MRSA NCTC 10442. Resulting rDNA profiles could be differentiated on the basis of 22 patterns which were unaffected by changes in plasmid, transposon, enterotoxin A or phage content. Percentage similarity values were calculated using the Dice coefficient and UPGA clustering. Australian and epidemic (EMRSA-1) isolates from the UK showed a high degree of similarity, but the pattern was not unique and was also found in MSSA and other MRSA, e.g. NCTC 10442. An MSSA of phage group II was the most distinct isolate. The method shows potential as an additional tool in a complex typing system, types non-phage typable strains and may provide clues to the clonal evolution of MRSA and MSSA.

Analysis of epidemic and sporadic strains of Listeria monocytogenes by pyrolysis mass spectrometry

Twenty-six cultures of Listeria monocytogenes, serovar 4b, including 10 from a food-borne outbreak in Switzerland and sporadic patient and food isolates from both Switzerland and the UK, were compared by pyrolysis mass spectrometry (PMS). This clustered all of the Swiss epidemic isolates with four other isolates indistinguishable from the Swiss strain by phage typing, a phage non-typable isolate from a Swiss patient not known to be part of the epidemic and two strains (both from Switzerland) of a different phage type. The eight strains excluded from the PMS-derived cluster were all either known to be unrelated to the epidemic, or of a phage type distinct from the epidemic strain, or both.

RRNA probing of chromosomal DNA of epidemic and sporadic isolates of Salmonella enterica subsp. Enterica serovar kottbus from Northern and Southern Italy

Fifty-two strains of Salmonella enterica subsp. enterica serovar Kottbus, identified at the Centres of Enterobacteriaceae of Northern and Southern Italy, were investigated by molecular genetic methods. Thirteen isolates were recovered during two food-poisoning outbreaks that occurred in May 1987 in Lombardy. The rDNA gene restriction patterns, obtained by probing endonuclease cleaved chromosomal DNA with photobiotin labeled Escherichia coli rRNA, revealed some heterogeneity among strains isolated from Southern Italy, whereas Northern Italy isolates exhibited virtually identical banding patterns.

Persistent colonization of carbon dioxide incubators withCandida parapsilosis

Recurrent contamination of bacteriological specimens with Candida parapsilosis led to epidemiological investigations which indicated persistent colonization of carbon dioxide incubators as the most likely source. Changes in the technical arrangements and institution of a meticulous cleansing protocol eliminated contamination of specimens but not colonization of the incubators. Tests for tolerance of 17% NaCl and survival at 50 degrees C, and SDS-PAGE analysis of crude cell extracts allowed discrimination between epidemic and non-epidemic isolates, while enzyme profile analysis and susceptibility studies failed as typing methods.

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).

Plasmid composition and virulence-associated factors of Yersinia pestis isolates from a plague outbreak at the Paraiba State, Brazil.

Pathogenic Yersinia pestis isolates were collected during a plague outbreak at the Paraiba State in 1986. The Y. pestis isolates were investigated for the presence of virulence-associated factors and plasmid content. All strains analysed were proficient in the expression of the VW and fraction 1 antigens, pigment adsorption and pesticin-fibrinolysin-coagulase production. A similar plasmid profile composed by four plasmid with molecular weight of 60, 44, 14.9, and 6.4 Megadaltons (MD) was found in all strains. DNA cleavage with EcoRI restriction enzyme further demonstrated the uniform plasmid content of the Y. pestis isolates. Seven additional Y. pestis strains, previously isolated in the same region but in an endemic state, showed the same plasmid fingerprint. The lack of any detectable difference between epidemic and endemic isolates as well as the value of plasmid fingerprints in epidemiology of Y. pestis is discussed.

Recombinant alpha and beta interferous inhibit epidemic isolates of acute hemorrhagic conjunctivities virus : M.R. Langford, and M. Yin-Murphy. Department of Ophthamology, Louisiana State University Medical School, Shreveport, LA and Department of Microbiology, National University of Singapore, Singapore

Recombinant alpha and beta interferous inhibit epidemic isolates of acute hemorrhagic conjunctivities virus : M.R. Langford, and M. Yin-Murphy. Department of Ophthamology, Louisiana State University Medical School, Shreveport, LA and Department of Microbiology, National University of Singapore, Singapore

Comparison of biochemical and serological typing results and antimicrobial susceptibility patterns in the epidemiological investigation of Klebsiella spp

An analysis of the serological and biochemical typing results of 925 clinical isolates of klebsiella revealed that biotyping and serotyping of klebsiella could replace each other for epidemiological purposes. The combination of both typing methods provided even more epidemiological information in analysing clusters of particular serotypes and biotypes in time. Clustering serotypes, mainly of neonatal origin, were nearly uniformly more resistant to the antibiotics in common use than other serotypes. Biotyping as well as serotyping of klebsiella isolates recovered from environmental surveys in the neonatal ward showed that epidemic and non-epidemic klebsiella isolates could occasionally be cultured from the environment and from the staff.

Plasmid profiles of multi-resistant Staphylococcus aureus at a children's hospital.

Twenty-six strains of multi-resistant Staphylococcus aureus (MRSA) recovered from November 1985 to February 1986 were screened for their plasmid profiles. Among them, 11 strains were recovered from 8 patients with definite nosocomial infections. The results showed that one MRSA strain carrying 5 plasmids with molecular weight ranging from 1.75 X 10(6) to 5 X 10(6) daltons was epidemic in our hospital while 3 other distinct strains caused cross infections in both the burn ward and the medical wards. The epidemic strain and two other hospital-acquired strains were found in identical form in the community, suggesting that the pattern of nosocomial MRSA can be affected by strains outside hospital. The plasmid profile analysis was employed to differentiate the epidemic isolates from the coisolates and to study the relation between clinical isolates. The method is apparently superior to antibiogram.

Disease severity‐related antigenic differences in dengue 2 strains detected by dengue 4 monoclonal antibodies

Low-passage epidemic Thai isolates of dengue 2 (DEN-2) were distinguished on the basis of associated disease severity by detection of virus determinants involved in enhanced macrophage infection when incubated with a panel of monoclonal antibodies derived from a DEN-4 strain associated with grade II dengue hemorrhagic fever (DHF). DEN-2 strains associated with DHF typically had multiple determinants that were involved in antibody-dependent enhancement of infection, whereas those associated with uncomplicated disease had few. These data provide further evidence that in epidemic areas where DHF is associated with prior circulation of low-level monotypic antibody, severe dengue disease could represent antibody-enhanced infection of human monocytes/macrophages.

Application of a technique for serogrouping Clostridium difficite in an outbreak of antibiotic-associated diarrhoea

A severe outbreak of Clostridium difficile antibiotic-associated diarrhoea (AAD) in an orthopaedic surgical unit is reported. Thirty-seven cases and eight relapses were observed. The 45 related strains together with another 13 strains of C. difficile isolated during the same period in other wards of the same hospital were typed by detection of cytotoxin production, determination of sorbitol fermentation and serogrouping by agglutination with six rabbit antisera defining the serogroups A, B, C, D, F and G. All the strains from the outbreak belonged to serogroup C, were toxigenic and fermented sorbitol. In contrast, four different patterns were observed in seven isolates from cases of AAD in other wards. Finally, six strains isolated from four asymptomatic adults, one adult suffering from shigellosis and one child with salmonellosis demonstrated two patterns different from that of the epidemic isolates. The data strongly suggest nosocomial spread of this micro-organism.

A community-based outbreak of infection with penicillin-resistant Neisseria gonorrhoeae not producing penicillinase (chromosomally mediated resistance).

From February through November of 1983, 199 cases of penicillin-resistant Neisseria gonorrhoeae infection were identified in a localized epidemic in Durham, North Carolina. The isolates did not produce beta-lactamase but were unusually resistant to penicillin (minimum inhibitory concentration, 2.0 to 4.0 micrograms per milliliter), and 15 of 16 patients treated with 4.8 million units of penicillin G procaine plus 1.0 g of probenecid did not respond to therapy. Recognition of the outbreak was impeded by a lack of routine surveillance for resistance other than that mediated by beta-lactamase. All epidemic isolates had a single serotype, auxotype, and antibiotic-susceptibility profile. The outbreak was halted by changing the treatment for all patients and their contacts to spectinomycin, and by intensive epidemiologic case-finding efforts. The emergence of such resistant strains poses potential major public health problems and indicates a need for reassessment of current surveillance procedures.

Genetic transfer of antimicrobial resistance and enterotoxigenicity among Escherichia coli strains.

To understand the role of enterotoxin (Ent) plasmids in epidemics of enterotoxigenic (ET) Escherichia coli diarrhea in the United States, we studied the genetics of Ent plasmids in relation to E. coli serotypes and R plasmids. Twenty-nine ET E. coli strains, including all epidemic isolates available at the Centers for Disease Control, Atlanta, Ga. (CDC), were assessed for the ability to transfer antimicrobial resistances (if present) by conjugation, to mobilize a nonconjugative R plasmid, and to cotransfer enterotoxigenicity with R determinants. Of the 12 ET E. coli strains isolated in the United States, 5 were able to transfer R plasmids; one strain cotransferred detectable enterotoxigenicity. Another four U.S. isolates were able to mobilize plasmid DNA, but no toxin production was detected in transconjugants. Of 17 resistant ET E. coli from South Asia, 13 were able to transfer R plasmids; 5 of those 13 cotransferred detectable Ent plasmids. In all, 22 ET E. coli strains (76%) were able to initiate conjugation and genetic transfers. Six of these strains (20%) were able to cotransfer enterotoxigenicity with a conjugative R plasmid at a detectable frequency. One of the six strains transferred R and Ent determinants on a single plasmid. These data are addressed in relation to the observed immobility of Ent and R during outbreaks of ET E. coli, the efficacy of prophylactic tetracycline, and the worldwide occurrence of a limited number of ET E. coli serotypes.

Molecular variation of type 1 vaccine-related and wild polioviruses during replication in humans

Molecular variation in clinical isolates of type 1 poliovirus was followed at the level of the genome by oligonucleotide mapping and at the level of the gene products by polyacrylamide gel electrophoresis of the viral proteins. Oligonucleotide maps of epidemiologically distinct isolates were very different. In contrast, isolates from the recent Netherlands-Canada-USA poliomyelitis epidemic displayed very similar maps. Small but reproducible map changes were consistently observed for consecutive case isolates, which revealed the continual mutation and selection of the epidemic virus during replication in humans. Direct comparison of the maps of the first (Netherlands, 5/78) and the last (USA 6/79) epidemic case isolates suggested that about 100 base changes were fixed into the average viral genome during 13 months of epidemic transmission. Isolates from two recent vaccinees and one epidemiologically unrecognized contact similarly revealed small map differences from each other and from the pattern characteristic of the type 1 oral vaccine. Accompanying the genome changes were alterations in the mobilities and cleavage rates of the viral proteins. For both vaccine-related and wild isolates, the greatest variabilities were detected in the virion surface proteins, a pattern of change likely to lead to antigenic drift of the virus. Oligonucleotide mapping offers an independent and definitive approach for the recognition of genetic and epidemiologic relationships between poliovirus isolates.