Strain Typing Data Research Articles

Listeria environmental sampling tests are compatible with polymorphic locus sequence typing.

Food processors invest significant resources into environmental sampling to detect contamination with potential pathogens, particularly Listeria monocytogenes. To facilitate these efforts, multiple environmental sampling tests (ESTs) have been developed and commercialized that minimize workload, turnaround time, and cost while providing convenient colorimetric detection. For presumptive-positive ESTs, we hypothesized that a relatively minor additional investment could provide, in addition to species confirmation, valuable strain typing data for tracking pathogen spread through a facility, identifying harborage sites, and distinguishing sporadic from persistent or resident contaminants. This hypothesis is based on the demonstrated compatibility of polymorphic locus sequence typing (PLST) with crude samples including food enrichments. Five Listeria ESTs were tested here: broth-based InSite (Hygiena), Path-Chek (Mericon), and Pathfinder (Hardy Diagnositics); and gel-based Petrifilm (3M) and HardyChrom (Hardy Diagnostics). ESTs were inoculated with strains representing two common L. monocytogenes serotypes and nonpathogenic Listeria innocua. Following incubation, broths or suspended colonies were heat treated to inactivate bacteria. Lysates or purified DNAs were prepared and used as templates in PCRs targeting the previously described PLST loci LmiMT1 and LisMT2. Single clear products were obtained from all inoculated ESTs; uninoculated controls were negative. PCR products were subjected to Sanger sequencing, yielding high-quality chromatograms. Phylogenetic analysis confirmed identities to previously determined sequences and revealed relatedness to serotype-matched strains represented in GenBank databases. PRACTICAL APPLICATION: Multiple environmental sampling tests have been commercialized in recent years to facilitate the proactive detection of pathogens, particularly Listeria monocytogenes, within food processing facilities. Coupling a positive detection test with strain typing would enhance its value by providing data that can be used to track pathogen spread through a facility, identify harborage sites, and distinguish sporadic from resident contamination.

Understanding Tuberculosis Transmission in the United Kingdom: Findings From 6 Years of Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats Strain Typing, 2010-2015.

Genotyping provides the opportunity to better understand tuberculosis (TB) transmission. We utilized strain typing data to assess trends in the proportion of clustering and identify the characteristics of individuals and clusters associated with recent United Kingdom (UK) transmission. In this retrospective cohort analysis, we included all culture-confirmed strain-typed TB notifications from the UK between 2010 and 2015 to estimate the proportion of patients that clustered over time. We explored the characteristics of patients in a cluster using multivariable logistic regression. Overall, 58.5% of TB patients were concentrated in 2,701 clusters. The proportion of patients in a cluster decreased between 2010 (58.7%) and 2015 (55.3%) (P = 0.001). Being a clustered patient was associated with being male and UK-born, having pulmonary disease, having a previous TB diagnosis, and having a history of drug misuse or imprisonment. Our results suggest that TB transmission in the UK decreased between 2010 and 2015, during which time TB incidence also decreased. Targeted cluster investigation and extended contact tracing should be aimed at persons at risk of being in a transmission chain, including UK-born individuals with social risk factors in clusters with a high proportion of patients having pulmonary disease.

Transmission events revealed in tuberculosis contact investigations in London

Contact tracing is a key part of tuberculosis prevention and care, aiming to hasten diagnosis and prevent transmission. The proportion of case-contact pairs for which recent transmission occurred and the typical timespans between the index case and their contact accessing care are not known; we aimed to calculate these. We analysed individual-level TB contact tracing data, collected in London from 20/01/2011-31/12/2015, linked to tuberculosis surveillance and MIRU-VNTR 24-locus strain-typing information. Of pairs of index cases and contacts diagnosed with active tuberculosis, 85/314 (27%) had strain typing data available for both. Of these pairs, 79% (67/85) shared indistinguishable isolates, implying probable recent transmission. Of pairs in which both contact and the index case had a social risk factor, 11/11 (100%) shared indistinguishable isolates, compared to 55/75 (75%) of pairs in which neither had a social risk factor (P = 0.06). The median time interval between the index case and their contact accessing care was 42 days (IQR: 16, 96). As over 20% of pairs did probably not involve recent transmission between index case and contact, the effectiveness of contact tracing is not necessarily limited to those circumstances where the index case has transmitted disease to their close contacts.

Phylogenetic inference of Coxiella burnetii by 16S rRNA gene sequencing.

Coxiella burnetii is a human pathogen that causes the serious zoonotic disease Q fever. It is ubiquitous in the environment and due to its wide host range, long-range dispersal potential and classification as a bioterrorism agent, this microorganism is considered an HHS Select Agent. In the event of an outbreak or intentional release, laboratory strain typing methods can contribute to epidemiological investigations, law enforcement investigation and the public health response by providing critical information about the relatedness between C. burnetii isolates collected from different sources. Laboratory cultivation of C. burnetii is both time-consuming and challenging. Availability of strain collections is often limited and while several strain typing methods have been described over the years, a true gold-standard method is still elusive. Building upon epidemiological knowledge from limited, historical strain collections and typing data is essential to more accurately infer C. burnetii phylogeny. Harmonization of auspicious high-resolution laboratory typing techniques is critical to support epidemiological and law enforcement investigation. The single nucleotide polymorphism (SNP) -based genotyping approach offers simplicity, rapidity and robustness. Herein, we demonstrate SNPs identified within 16S rRNA gene sequences can differentiate C. burnetii strains. Using this method, 55 isolates were assigned to six groups based on six polymorphisms. These 16S rRNA SNP-based genotyping results were largely congruent with those obtained by analyzing restriction-endonuclease (RE)-digested DNA separated by SDS-PAGE and by the high-resolution approach based on SNPs within multispacer sequence typing (MST) loci. The SNPs identified within the 16S rRNA gene can be used as targets for the development of additional SNP-based genotyping assays for C. burnetii.

Clonal replacement and expansion among invasive meningococcal isolates of serogroup W in France

Neisseria meningitidis group W (NmW) belonging to the clonal complex ST-11 (NmW/cc11) spread in Europe and in France in 2000 and declined thereafter. In France, invasive meningococcal disease (IMD) due to NmW increased again in 2012 and thereafter since 2015. Several sub-lineages of NmW/cc11 are circulating worldwide with successive epidemic waves. We aimed to describe recent epidemiological trends of NmW in France and to explore the microbiological and epidemiological characteristics associated with different NmW/cc11 sub-lineages. The epidemiology of NmW was described based on data collected through mandatory notification of IMD and strain typing data for culture-confirmed and PCR-confirmed cases for the period 2000-2016. All culture-confirmed cases due to NmW from the period 2010-2016 were characterised by whole genome sequencing (WGS). A detailed epidemiological analysis was performed for culture-confirmed cases on the basis of WGS data. During the period 2010-2016, genotyping was obtained for 148 cases including all the 132 culture-confirmed cases, among which 127 were matched with epidemiological data, and 16 PCR-confirmed cases (out of a total of 47 PCR-confirmed cases). An increase in IMD was observed in 2012 and was linked to isolates belonging to the "Anglo-French-Hajj" sub-lineage. These isolates have decreased significantly since 2013 and have been replaced by NmW/cc11 isolates related to the "South American - UK" sub-lineage which caused a marked increase in the number of cases of NmW in 2016. In this sub-lineage, the "original UK strain" was first detected in 2012 and increased thereafter, followed by the recently described "UK 2013-strain". Isolates related to the "South American-UK" sub-lineage represented 45% of all NmW cultured isolates from the whole period 2010-2016 but were the most frequent isolates in 2016, representing 76% of the total NmW typed isolates and 94% of the typed NmW/cc11 isolates. A changing pattern in the epidemiology of NmW has been observed in 2015-2016 in relation to the spread of the "UK 2013-strain" with a sharp increase in the number of cases among persons aged 15 years and over and a high case fatality rate (CFR). Among cases due to the "UK 2013-strain", 94% of cases were aged 15 years and over and the CFR was 28%. Our data suggest a recent clonal replacement among NmW/cc11 isolates with the expansion of the "South American-UK" sub-lineage in France and particularly the "UK 2013-strain" which was predominant in 2015 and 2016. A shift in the age-distribution of IMD due to NmW to older ages and the high CFR are consistent with the expansion of a new virulent clone in a naive population. These data may have an impact on tailoring vaccination strategies against NmW.

Recent household transmission of tuberculosis in England, 2010\u20132012: retrospective national cohort study combining epidemiological and molecular strain typing data

BackgroundWe estimate the proportion of tuberculosis (TB) in England due to recent household transmission, identify factors associated with being a household transmitter, and investigate the impact that identification of a case has on time to treatment of subsequent cases.MethodsTB cases notified between 2010 and 2012 in England in the same household as another case were identified; 24 locus MIRU-VNTR strain typing (ST) was used to identify household cases with likely recent transmission. Treatment delay in index and subsequent cases was compared. Risk factors for being a household transmitter were identified in univariable and multivariable analyses.ResultsOverall, 7.7% (1849/24,060) of TB cases lived in a household with another case. We estimate that 3.9% were due to recent household transmission. ST data was unavailable for 67% (1242) of household pairs. For those with ST data, 64% (386) had confirmed, 11% probable (66) and 25% (155) refuted household transmission. The median treatment delay was 65 days for index cases and 37 days for subsequent asymptomatic cases. Risk factors for being a household transmitter included being under 25 years old, UK-born with Black African, Indian or Pakistani ethnicity, or born in Somalia or Romania.ConclusionsThis study has a number of implications for household TB contact tracing in low incidence countries, including the potential to reduce the diagnostic delay for subsequent household cases and the benefit of using ST to identify when to conduct source contact tracing outside the household. As 25% of TB cases in households had discordant strains, households with multiple TB cases do not necessarily represent household transmission. The additional fact that 25% of index cases within households only had extra-pulmonary TB demonstrates that, if household contact tracing is limited to pulmonary TB cases (as recently recommended in UK guidelines), additional cases of active TB in households will be missed. Our finding that no lineage of TB was associated with recent household transmission and with no increased transmissibility in the Beijing lineage compared to others, suggests that the lineage need not impact contact tracing efforts. Improvements in contact tracing have the potential to reduce transmission of TB in low incidence countries.

Multiple large clusters of tuberculosis in London: a cross-sectional analysis of molecular and spatial data.

Large outbreaks of tuberculosis (TB) represent a particular threat to disease control because they reflect multiple instances of active transmission. The extent to which long chains of transmission contribute to high TB incidence in London is unknown. We aimed to estimate the contribution of large clusters to the burden of TB in London and identify risk factors.We identified TB patients resident in London notified between 2010 and 2014, and used 24-locus mycobacterial interspersed repetitive units–variable number tandem repeat strain typing data to classify cases according to molecular cluster size. We used spatial scan statistics to test for spatial clustering and analysed risk factors through multinomial logistic regression.TB isolates from 7458 patients were included in the analysis. There were 20 large molecular clusters (with n>20 cases), comprising 795 (11%) of all cases; 18 (90%) large clusters exhibited significant spatial clustering. Cases in large clusters were more likely to be UK born (adjusted odds ratio 2.93, 95% CI 2.28–3.77), of black-Caribbean ethnicity (adjusted odds ratio 3.64, 95% CI 2.23–5.94) and have multiple social risk factors (adjusted odds ratio 3.75, 95% CI 1.96–7.16).Large clusters of cases contribute substantially to the burden of TB in London. Targeting interventions such as screening in deprived areas and social risk groups, including those of black ethnicities and born in the UK, should be a priority for reducing transmission.

TB outbreak investigation in a faith based boarding school: Challenges and control measures

Background: Tuberculosis burden in UK is unacceptably high compared to similar countries. TB outbreaks in schools are extremely complex and present public health challenges. 7 cases of TB were notified at a faith based female boarding school in a UK city over 5 years. We aim to describe this challenging outbreak investigation. Methods & Materials: In 2008, the first pulmonary TB case was notified to public health authorities. In subsequent years, more cases were reported. Epidemiological investigations were undertaken in school including risk assessment, contact tracing and screening following each case to identify cases of active or latent TB. Mass screening at school was extremely challenging both in terms of logistics and managing cultural sensitivities. Initially, TB strain typing testing was not available. In 2013, following a case, the whole school was screened again. In early 2014, retrospective review and 24 loci TB strain typing was done to identify transmission chains. Results: From 2008 to 2013, mass TB screening was done four times in this school. In total, 1524 students and staff were screened. Of these, 98 had latent and 13 had active TB. In 2012, 64 cases (11%) had latent TB and 9 (1.6%) had active TB. Epidemiological investigations did not reveal any chains of transmission. TB strain typing in 2014 revealed that the 2012 case had identical 24 loci strain typing to the 2008 case. Strain typing was not possible in extra-pulmonary TB cases, and cases with missing loci could not be linked microbiologically. Conclusion: Strain typing data suggests ongoing TB transmission in school. Finding latent and active TB cases in school does not indicate exposure to notified cases, as most students are from a high risk population group. Whole genome sequencing could provide accurate data on ‘lineages’ of M. tuberculosis for evidence on chains of transmission. This outbreak highlights the need for innovative TB prevention and control strategies in such settings. Control measures included risk assessment and screening of new students, BCG vaccination history, prompt referral of symptomatic cases to TB services and raising awareness about TB. Proactive control measures in such high risk settings can minimise spread and prevent future outbreaks.

P91 Incorporating tuberculosis strain typing data into routine contact tracing investigations: experience from the field

Strain typing of tuberculosis (TB) isolates by 24 loci mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) is now a routine laboratory tool for TB control, but its utility in...

Molecular and phenotypic evidence for the spread of three major methicillin-resistant Staphylococcus aureus clones associated with two characteristic antimicrobial resistance profiles in China

The distribution of methicillin-resistant Staphylococcus aureus (MRSA) clones is dynamic and geographically unique. To understand the changing epidemiology of MRSA infections in China, we performed a prospective, multicity surveillance study with molecular typing and phenotypic analysis to determine the association of major prevalent clones with their antimicrobial resistance profiles. A total of 517 S. aureus isolates collected between January 2009 and March 2012 from six cities in China were subjected to antibiogram analysis and molecular typing, including staphylococcal cassette chromosome mec typing, multilocus sequence typing, staphylococcal protein A gene typing and PFGE typing. Among the isolates collected, 309 were characterized as MRSA, with a prevalence of 59.8%. Three major clones were found to be prevalent in China: ST239-MRSA-III-t030, ST239-MRSA-III-t037 and ST5-MRSA-II-t002. These three clones were associated with two characteristic resistance profiles, namely, gentamicin/ciprofloxacin/rifampicin/levofloxacin for the first clone and gentamicin/ciprofloxacin/clindamycin/erythromycin/tetracycline/levofloxacin/trimethoprim/sulfamethoxazole for the latter two. Several geographically unique minor clones were also identified. The predominant MRSA clones in China were associated with characteristic antimicrobial resistance profiles. Antibiotics for treating patients with MRSA infections can be selected based on the strain typing data.

Ethnicity and mycobacterial lineage as determinants of tuberculosis disease phenotype

BackgroundEmerging evidence suggests that Mycobacterium tuberculosis (Mtb) lineage and host ethnicity can determine tuberculosis (TB) clinical disease patterns but their relative importance and interaction are unknown.MethodsWe evaluated prospectively collected TB...

Treatment of Tuberculosis in a Region with High Drug Resistance: Outcomes, Drug Resistance Amplification and Re-Infection

IntroductionEmerging antituberculosis drug resistance is a serious threat for tuberculosis (TB) control, especially in Eastern European countries.MethodsWe combined drug susceptibility results and molecular strain typing data with treatment outcome reports to assess the influence of drug resistance on TB treatment outcomes in a prospective cohort of patients from Abkhazia (Georgia). Patients received individualized treatment regimens based on drug susceptibility testing (DST) results. Definitions for antituberculosis drug resistance and treatment outcomes were in line with current WHO recommendations. First and second line DST, and molecular typing were performed in a supranational laboratory for Mycobacterium tuberculosis (MTB) strains from consecutive sputum smear-positive TB patients at baseline and during treatment.ResultsAt baseline, MTB strains were fully drug-susceptible in 189/326 (58.0%) of patients. Resistance to at least H or R (PDR-TB) and multidrug-resistance (MDR-TB) were found in 69/326 (21.2%) and 68/326 (20.9%) of strains, respectively. Three MDR-TB strains were also extensively resistant (XDR-TB). During treatment, 3/189 (1.6%) fully susceptible patients at baseline were re-infected with a MDR-TB strain and 2/58 (3.4%) PDR-TB patients became MDR-TB due to resistance amplification. 5/47 (10.6%) MDR- patients became XDR-TB during treatment. Treatment success was observed in 161/189 (85.2%), 54/69 (78.3%) and 22/68 (32.3%) of patients with fully drug susceptible, PDR- and MDR-TB, respectively. Development of ofloxacin resistance was significantly associated with a negative treatment outcome.ConclusionIn Abkhazia, a region with high prevalence of drug resistant TB, the use of individualized MDR-TB treatment regimens resulted in poor treatment outcomes and XDR-TB amplification. Nosocomial transmission of MDR-TB emphasizes the importance of infection control in hospitals.

Evolution and Spread of a Multidrug-Resistant Proteus mirabilis Clone with Chromosomal AmpC-Type Cephalosporinases in Europe

Proteus mirabilis isolates obtained in 1999 to 2008 from three European countries were analyzed; all carried chromosomal AmpC-type cephalosporinase bla(CMY) genes from a Citrobacter freundii origin (bla(CMY-2)-like genes). Isolates from Poland harbored several bla(CMY) genes (bla(CMY-4), bla(CMY-12), bla(CMY-14), bla(CMY-15), and bla(CMY-38) and the new gene bla(CMY-45)), while isolates from Italy and Greece harbored bla(CMY-16) only. Earlier isolates with bla(CMY-4) or bla(CMY-12), recovered in France from Greek and Algerian patients, were also studied. All isolates showed striking similarities. Their bla(CMY) genes resided within ISEcp1 transposition modules, named Tn6093, characterized by a 110-bp distance between ISEcp1 and bla(CMY), and identical fragments of both C. freundii DNA and a ColE1-type plasmid backbone. Moreover, these modules were inserted into the same chromosomal site, within the pepQ gene. Since ColE1 plasmids carrying ISEcp1 with similar C. freundii DNA fragments (Tn6114) had been identified earlier, it is likely that a similar molecule had mediated at some stage this DNA transfer between C. freundii and P. mirabilis. In addition, isolates with bla(CMY-12), bla(CMY-15), and bla(CMY-38) genes harbored a second bla(CMY) copy within a shorter ISEcp1 module (Tn6113), always inserted downstream of the ppiD gene. Sequence analysis of all mobile bla(CMY-2)-like genes indicated that those integrated in the P. mirabilis chromosome form a distinct cluster that may have evolved by the stepwise accumulation of mutations. All of these observations, coupled to strain typing data, suggest that the bla(CMY) genes studied here may have originated from a single ISEcp1-mediated mobilization-transfer-integration process, followed by the spread and evolution of a P. mirabilis clone over time and a large geographic area.

Use of Strain Typing Data to Estimate Bacterial Transmission Rates in Healthcare Settings

To create an affordable and accurate method for continuously monitoring bacterial transmission rates in healthcare settings. We present a discrete simulation model that relies on the relationship between in-hospital transmission rates and strain diversity. We also present a proof of concept application of this model to a prospective molecular epidemiology data set to estimate transmission rates for Pseudomonas aeruginosa and Staphylococcus aureus. Inpatient units of an academic referral center. All inpatients with nosocomial infections. Mathematical model to estimate transmission rates. Maximum likelihood estimates for transmission rates of these two species on different hospital units ranged from 0 to 0.36 transmission event per colonized patient per day. This approach is feasible, although estimates of transmission rates based solely on strain typed clinical cultures may be too imprecise for routine use in infection control. A modest level of surveillance sampling substantially improves the estimation accuracy.

Nucleotide sequence analysis of Plum pox virus isolate W3174: Evidence of a new strain

The nucleotide sequence of Plum pox virus (PPV) isolate W3174 was determined. The virus genome consists of 9788 nucleotides (nt), excluding the poly(A) tail at the 3′-terminus, with 5′- and 3′-untranslated regions (UTRs) of 146 and 219 nt, respectively. The deduced polyprotein consists of 3141 amino acid (aa) residues, with the coat protein region consisting of 993 nt (331 aa). In comparisons with PPV-D, -M, -EA and -C isolates, nucleotide identity levels ranged from 79 to 80% for the entire genome and from 78 to 79%, 78 to 81%, and 92 to 95% for the NIb, CP, and 3′-UTR regions, respectively. The majority of nucleotide substitutions in the NIb region of W3174 are silent, while substitutions in the CP region are not silent, giving aa identities of 89–91% and 79–81%, respectively. The HC-Pro region contains the KITC and PTK motifs, and the DAG motif is located at positions 12–14 of the deduced CP aa sequence, all associated with aphid transmission. Phylogenetic analysis based on the complete genome sequence, the NIb, CP, and 3′-UTR region were performed. PPV-W3174 consistently formed a distinct clade or group, when compared to members of all four recognized strains of PPV, indicating that it is genetically distinct. These results are consistent with serological and nucleic acid-based strain typing data and justify recognition of this isolate as representative of a new strain identified as PPV-W.

Mastitis-Causing Streptococci Are Important Contributors to Bacterial Counts in Raw Bulk Tank Milk

The objective of this study was to probe the contribution of streptococci to the microbial quality of raw milk. Over a 5-month period, bulk tank milk samples from 48 New York State dairy farms were analyzed qualitatively for bacterial ecology and quantitatively for total bacterial, streptococcal, staphylococcal, and gram-negative bacterial counts. Linear regression analysis was used to determine the contribution of differential counts to total bacterial counts. Streptococci, staphylococci, and gram-negative bacteria accounted for 69, 3, and 3% of total bacterial count variability, respectively. Randomly selected Streptococcus isolates from each bulk tank milk sample were identified to species by means of the API 20 STREP identification system. The most commonly identified streptococcal species were Streptococcus uberis, Aerococcus viridans, and Streptococcus agalactiae, which were detected in 81, 50, and 31% of 48 bulk tank samples, respectively. For five herds, S. uberis isolates from bulk tank milk and individual cows were characterized by PvuII ribotyping. A farm-specific dominant ribotype was found in each bulk tank sample, and that ribotype was isolated from at least one cow within each herd of origin. Bacteriological and strain typing data indicate that control of streptococci, specifically mastitis-causing species, is important for improvement of the microbial quality of raw milk in New York State.

Molecular epidemiology of Mycobacterium bovis: exploiting molecular data

‘Molecular epidemiology’ is defined as the integration of conventional epidemiological approaches with molecular techniques to track specific strains of pathogens in order to understand the distribution of disease in populations. It has become a very powerful tool in the study of Mycobacterium tuberculosis and human tuberculosis, where it has been exploited to provide ‘added value’ to conventional epidemiological approaches (contact tracing) and has often challenged accepted dogmas. It has been used to confirm epidemiologically suspected transmission, to detect epidemiologically unsuspected transmission, to identify risk factors and environments where transmission is occurring, to detect laboratory errors and to monitor the efficacy of tuberculosis control programmes.For Mycobacterium bovis and bovine tuberculosis, molecular epidemiology has a key role to play in providing more precise epidemiological data on the issues of interbovine transmission and the role of wildlife reservoirs in disease maintenance and transmission. M. bovis strains may also differ in key biological properties, such as virulence, transmissibility, stability and antigenic variation, which may help to explain field observations. There may be correlation between strain type and ‘herd level’ factors such as breakdown size etc. Molecular ‘strain typing’ studies have provided useful information in several countries, notably New Zealand, where strain typing data is used as an integral part of M. bovis control schemes, to influence the level of herd testing or wildlife control and to define the extent and spread of infected wildlife.This presentation will review the methods and approaches currently appropriate for M. bovis strain typing and will review selected applications as well as discussing future perspectives and challenges for the application of molecular epidemiology to bovine tuberculosis.

Epidemiologic typing of methicillin-resistant Staphylococcus aureus in neonate intensive care units using pulsed-field gel electrophoresis.

To elucidate the mode of dissemination of methicillin-resistant Staphylococcus aureus (MRSA) in neonate intensive care units (NICUs), a total of 223 isolates from 3 separate hospitals were investigated between 1994 and 1996 by a DNA fingerprinting technique with pulsed-field gel electrophoresis (PFGE). Exoprotein profiles of some strains were also examined using SDS-polyacrylamide gel-electrophoresis (SDS-PAGE) and the assessment of enzyme/toxin production such as coagulase, enterotoxin and TSST-1. Judging from the strain typing data from PFGE results and the epidemiological data, 2 different types of PFGE patterns (A and B) and their subtypes (A', A'' and B') were identified. The A type including A' and A'' (comprising approximately 95% of the isolates) was markedly dominant. Only 5% of the isolates belonged to type B and subtype B'. On the other hand, MRSA isolated from adult patients admitted to the same hospital showed many different PFGE patterns. The results strongly suggested that some strain(s) with specific PFGE pattern(s) is prevalent in NICUs. Furthermore, isolates which expressed the same PFGE pattern did not always express the same SDS-PAGE pattern. There were some isolates with different abilities to produce coagulase, enterotoxin C and toxic-shock syndrome toxin (TSST)-1, and the abilities had no relation with a particular type of PFGE pattern. Therefore, a combination of PFGE analysis and biochemical analyses of coagulase, enterotoxin C and TSST-1 may provide us with more detailed information for the epidemiological study of MRSA in NICUs.

Amplification of the meningococcal porB gene for non-culture serotype characterization.

Since 1992, the proportion of culture-confirmed meningococcal infections compared with numbers of notified cases of meningococcal disease has decreased in England and Wales. As most meningococcal strain characterization methods depend on a clinical isolate, this has resulted in a loss of epidemiological information. To address this problem, and to aid nonculture diagnosis, a semi-nested PCR protocol for the amplification of the meningococcal porB gene from clinical specimens was developed. This gene encodes the meningococcal serotyping antigen; strain typing data was provided by hybridization of allele-specific oligonucleotide probes to the digoxigenin-labelled porB amplicon in a 'PCR ELISA'. This assay was specific for meningococcal DNA and sensitivities of 0.81 for cerebrospinal fluid (CSF), 0.57 for serum, and 0.62 for whole blood taken from patients with proven meningococcal infection were obtained.

Concept of segmentation in nosocomial epidemiology: Epidemiological relation among antimicrobial-resistant isolates of Pseudomonas aeruginosa

Typing studies on 271 clinical strains of Pseudomonas aeruginosa isolated from the University Teaching Hospital were conducted to obtain their serotypes, antimicrobial susceptibility patterns and plasmid profiles. These strain typing data were arranged through multivariate statistical analysis by computation to classify individual strains. Plots in the scatter diagrams obtained from both principal component analysis and quantification theory type III expressed the clinical strains of P. aeruginosa with various degrees of antimicrobial resistance. Epidemiological relation among these clinical strains was analysed in those scatter diagrams by segmentation, in combination with their epidemiological information (date and place of isolation, type of specimen, etc.). The results showed that the serotype E strains both with high-level resistance to gentamicin and with a plasmid of 3.9 x 10(6) dalton, and the strains resistant to more than five antimicrobial agents, were colonized and localized each in certain clinical wards for inpatients. It was suggested that segmentation analysis could be of practical use in the management of nosocomial infection control against P. aeruginosa with antimicrobial resistance.