Elizabeth Bannerman

Comparison of PCR-based DNA fingerprinting techniques for the identification of Listeria species and their use for atypical Listeria isolates.

Four PCR-based DNA fingerprinting techniques were compared for their ability to identify at the species level a heterogeneous collection of isolates belonging to the six valid Listeria species. 16S rDNA-RFLP analysis identified all species and 16S rDNA-SSCP analysis identified almost all species. Also, isolates with unusual biochemical characteristics and/or unusual antigenic composition could be identified correctly. rRNA-intracistronic length polymorphism analysis suffered from high intraspecific variability, a limited number of fragments per profile, and small length differences between the spacers of different species. tRNA-intergenic length polymorphism analysis resulted in identification of all isolates but one, when fluorescent DNA capillary electrophoresis was used such that fragment length differences of 1 bp could be resolved. The four techniques yielded comparable results relevant to the taxonomy of Listeria. They all indicate a high degree of genetic relatedness between L. innocua and L. welshimeri, homogeneity of L. grayi, distinct but clear relatedness of L. grayi to the other Listeria species, a clear distinction between the two subspecies of L. ivanovii, and a clear distinction between Listeria isolates and isolates from closely related taxa or from species which are phenotypically difficult to distinguish from Listeria. New sequence determination of the 16S rRNA gene was necessary to obtain sequences in accordance with the findings of 16S rDNA-RFLP analysis.

Typing Listeria monocytogenes: a comparison of random amplification of polymorphic DNA with 5 other methods

One hundred Listeria monocytogenes strains were typed by random amplification of polymorphic DNA (RAPD) with three different primers, and the results were compared with those obtained by serotyping, ribotyping, multilocus enzyme electrophoresis, restriction enzyme analysis and phage typing. The RAPD patterns of strains appear to be stable during epidemics even over periods of several years. Reproducibility of the RAPD patterns was good. The discriminatory power of RAPD typing was the best among all the methods tested. RAPD is therefore a very promising tool in the study of listeriosis epidemiology. However, the problems related to the standardization of the technique first have to be resolved before the wide use of RAPD is possible.

Serotyping of 80 strains from the WHO multicentre international typing study of Listeria monocytogenes

Serotyping was carried out on 80 coded strains, distribute to all laboratories taking part in the WHO L. monocytogenes multicenter subtyping study. All six laboratories used the method recommended by their coordinator. All 80 strains were typeable. There was complete agreement between the six laboratories on 49 (61.3%) strains (21 serovar 1/2a and 28 serovar 4b strains) which in turn were identical to the expected serovars, known only after decoding. The intralaboratory reproducibility carried out on 11 duplicate strains, ranged from 82 to 100%, with a medium value of 91%. Reproducibility of serotyping L. monocytogenes strains according to serovar varied from 33.3 to 100% for serotypes 3b and 1/2a, respectively, with serovar 4b (x) being incorrectly identified in all six laboratories. Serotyping of L. monocytogenes is easy and simple and is a useful prerequisite for other finer and more discriminatory typing methods. Problems may however, be encountered mainly with the flagellar antigenic factors. There is a need, therefore, for preparing antisera of good quality from which efficient antigenic factors can be obtained.

Characterization of Human Invasive Isolates of Listeria monocytogenes in Sweden 1986-2007

Since 1986, 68% of the Listeria monocytogenes isolates from human cases of invasive listeriosis in Sweden are available for retrospective studies. The aim of the present study was to characterize 601 human invasive isolates of L. monocytogenes in Sweden from 1986 to 2007 by using serotyping and pulsed-field gel electrophoresis. Since 1996, serovar 4b was permanently reduced to the second or third most common serovar in human cases in Sweden. During the latter period, 2000-2007, only 13% belonged to serovar 4b and 71% to 1/2a. The dendrogram, based on pulsovars, reveals two clusters with different serovars. Cluster 1 exhibits serovars 4b and 1/2b, whereas cluster 2 consists of serovar 1/2a. Serovar 1/2a seems to be more heterogeneous than serovar 4b.

Invasive disease and paediatric carriage of Streptococcus pneumoniae in Ghana

Abstract This study was carried out primarily to evaluate the public health burden related to Streptococcus pneumoniae in Ghana and to provide related preliminary molecular epidemiological data on the organism. Invasive and nasopharyngeal specimens were screened for S. pneumoniae, and isolates were subjected to serotyping, multilocus sequence typing (MLST) and antibiotic susceptibility testing. Overall, the prevalence of S. pneumoniae in cerebrospinal fluid (CSF) was 1.7%, in blood was 0.2%, and in nasopharyngeal specimens was 15.3%. The prevalence of multiple drug resistance among the isolates was 48.6%, while the percentage resistance to various drugs was in the range of 11.1–84.0%. Serotyping of the S. pneumoniae isolates showed 7 different serotypes (3, 6B, 9, 10, 14, 16 and 23F). The extent of coverage of serotypes by the 7-valent pneumococcal conjugate vaccine was 57.1%, for the 10-valent vaccine was 57.1%, and for the 13-valent vaccine was 71.4%. MLST of 7 housekeeping genes of the organism showed a high level of genetic diversity among the isolates. S. pneumoniae appears to be an important organism in invasive infections in Ghana, being the most prevalent organism in CSF in this study. The high multiple drug resistance of the organism observed heightens the public health burden, which may be controlled by pneumococcal conjugate vaccines to a large extent.

Typing of Listeria monocytogenes by monocin and phage receptors

One hundred strains of Listeria monocytogenes from both sporadic and epidemic cases were typed by monocin production combined with phage receptor and reverse phage receptor methods. The monocin-phage combination gave 72 types with 100% typability and 97% reproducibility. The results were compared to those of serotyping, phage typing, ribotyping, multilocus enzyme electrophoresis, restriction enzyme analysis and RAPD (random amplification of polymorphic DNA). The monocin/phage types were comparable in terms of discrimination with other methods for epidemiological investigations. The index of discrimination of using the monocin typing and phage receptor/reverse phage receptor method combination (0.99) for both the 87 epidemiologically unrelated strains and the epidemiologically important serogroup 4 strains was the highest of the seven different methods analysed. This combination of methods was simple, highly discriminatory and reproducible and can be carried out in a non-specialized laboratory. However, like most of the other Listeria typing methods, both the method and the indicator test strains need to be standardized.

Susceptibility Profiles of Mycobacterium ulcerans Isolates to Streptomycin and Rifampicin in Two Districts of the Eastern Region of Ghana

Background. Drug resistance is a major challenge in antibiotic chemotherapy. Assessing resistance profiles of pathogens constitutes an essential surveillance tool in the epidemiology and control of infectious diseases, including Buruli ulcer (BU) disease. With the successful definitive management of BU using rifampicin and streptomycin, little attention had been paid to monitoring emergence of resistant Mycobacterium ulcerans (M. ulcerans) isolates in endemic communities. This study investigated the susceptibility profiles of M. ulcerans isolates from two BU endemic areas in Ghana to streptomycin and rifampicin. Methods. The antibiotic susceptibility of seventy (70) M. ulcerans isolates to rifampicin and streptomycin was determined simultaneously at critical concentrations of 40 µg/mL and 4 µg/mL, respectively, by the Canetti proportion method. Results. Resistance to rifampicin was observed for 12 (17.1%) M. ulcerans isolates tested, whilst 2 (2.9%) showed resistance to streptomycin. None of the isolates tested showed dual resistance to both rifampicin and streptomycin. Conclusion. Outcomes from this study may not be reflective of all BU endemic communities; it, however, provides information on the resistance status of the isolates, which is useful for monitoring of M. ulcerans, as well as BU disease surveillance and control.

Evaluating decontamination protocols for the isolation of Mycobacterium ulcerans from swabs

BackgroundMycobacterium ulcerans (M. ulcerans) is the causative agent of Buruli Ulcer (BU) disease. In order to inhibit the growth of the microbial contaminants during culture of M. ulcerans, it is necessary to decontaminate BU samples with effective chemical agents. This study aimed at investigating some selected chemicals as potential decontamination agents for the isolation of M. ulcerans from swabs.ResultsPovidone iodine at 0.5 and 1% exhibited the lowest contamination and recovery rate for microbial contaminants and M. ulcerans. The most effective decontamination method was the protocol using 2% cetylpyridinium chloride/4% sodium chloride (recovery rate = 53%, contamination rate = 14%). The observed difference between the recovery rate of 2% CPC/4% NaC and the other protocols was however not statistically significant (p = 0.76).ConclusionsTwo percent (2%) cetylpyridinium chloride/4% sodium chloride can be conveniently used as an alternative decontamination method for the isolation of M. ulcerans from swabs.