- Volume 54, Issue 9, 2005

Eight identification methods were evaluated against 100 isolates of Neisseria gonorrhoeae and 21 non-gonococcal Neisseria strains. The methods examined included four commercial biochemical kits, API NH, RapID NH, Gonochek II and Neisseria Preformed Enzyme Test (PET), three immunological kits, Phadebact Monoclonal GC test, GonoGen II and MicroTrak, and one in-house carbohydrate-utilization method, cystine trypticase agar (CTA) sugars. The percentage of isolates unambiguously identified as N. gonorrhoeae by each of the methods was as follows: API NH, 66 %; RapID NH, 64 %; GonoChek II, 66 %; Neisseria PET, 66 %; Phadebact Monoclonal GC OMNI test, 99 %; GonoGen II, 100 %; MicroTrak, 100 %; and CTA sugars, 96 %. The low sensitivity of the biochemical kits for the identification of N. gonorrhoeae was due to a lack of the enzyme proline iminopeptidase (Pip) in 34 % of the isolates examined. All the biochemical kits utilized the presence of this enzyme as a marker for N. gonorrhoeae. The Phadebact Monoclonal GC kit, GonoGen II, MicroTrak, CTA sugars and the API NH kit all exhibited high specificity, but non-gonococcal Neisseria were misidentified as N. gonorrhoeae using RapID NH (two strains), Gonochek II (11 strains) and Neisseria PET (11 strains). Whilst the isolates examined in this study may not be truly representative, they do indicate that N. gonorrhoeae isolates lacking the enzyme Pip can give anomalous results when using commercially available biochemical tests and that some non-pathogenic Neisseria species are still being misidentified using some biochemical kits. This further reinforces the recommendation that any dubious biochemical result should be confirmed with an immunological test.

Mediterranean spotted fever (MSF) is a tick-borne rickettsiosis caused by ‘Rickettsia conorii complex’ strains. In Portugal, R. conorii and Israeli tick typhus (ITT) are the aetiological agents of this disease. A novel 65 bp tandem repeat was identified by the analysis of the R. conorii Malish 7 whole genome sequence with an appropriate algorithm for searching for repeated sequences. The variable number tandem repeat (VNTR) was named VNTR Rc-65 and this locus was amplified by PCR and sequenced in order to characterize the repeat diversity within different rickettsial strains including Portuguese strains isolated from clinical and vector samples. The VNTR Rc-65 has seven alleles within the rickettsial strains studied and a diversity index value of 0.71, meaning that this locus has a great discriminatory capacity and therefore can be used for identification of closely related strains. PCR amplification of the Rc-65 locus can be used to differentiate between the Portuguese R. conorii Malish-like and Israeli tick typhus strains, enabling a more accurate and rapid identification of these rickettsial isolates.

A membrane-filter-based, fluorescent Gram stain method for bacterial detection in cerebrospinal fluid samples was developed and evaluated as a rapid, sensitive alternative to standard Gram stain protocols. A recently developed, modified version of the aluminium oxide membrane Anopore with low-fluorescence optical properties showed superior performance in this application. Other aspects of the fluorescent Gram stain system that were evaluated include membrane filter selection, strategies to reduce fluorescence fading and the effect of patient blood cells on bacterial detection in the fluorescently stained cerebrospinal fluid samples. The combination of the membrane filter's bacteria-concentrating ability and absolute retention along with high-contrast, fluorescent Gram discriminating dyes enabled rapid bacterial detection and Gram discrimination, with a 1–1.5 order of magnitude increase in the bacterial concentration limit of detection.

In the present study, it was shown that repetitive-DNA-element PCR fingerprinting using the (GTG)5 primer [(GTG)5-PCR] is a rapid and reliable tool to genotypically differentiate members of the recently described pan-European multi-resistant Acinetobacter baumannii (MAB) clone III from the known MAB clones I and II. The identification of four new representatives of the MAB clone III dating from 1991 to 1993 by (GTG)5-PCR indicates that this clone has persisted in European hospitals since the beginning of the 1990s. Tetracycline (TET) resistance was found to be common among clone III strains, including one strain that also displayed resistance to minocycline. The TET-resistance phenotype in this MAB clone appeared to be strongly associated with the presence of the efflux-type gene tet(A), but the fact that some members lack this gene or have acquired an additional tet gene [i.e. tet(M)] suggests that the tet gene carriage in the pan-European clone III population may have diversified in time and space. In contrast, all clone III strains shared the previously described aminoglycoside resistotype encoded by the aminoglycoside-modifying genes aphA6 and the class 1 integron-associated aadB, which may point to the fact that these genes probably are more stably inherited in MAB clone III compared to tet genes.

The near full-length sequences of the groESL genes were determined and analysed among eight reference strains (serotypes a to h) representing five species of mutans group streptococci. The groES sequences from these reference strains revealed that there are two lengths (285 and 288 bp) in the five species. The intergenic spacer between groES and groEL appears to be a unique marker for species, with a variable size (ranging from 111 to 310 bp) and sequence. Phylogenetic analysis of groES and groEL separated the eight serotypes into two major clusters. Strains of serotypes b, c, e and f were highly related and had groES gene sequences of the same length, 288 bp, while strains of serotypes a, d, g and h were also closely related and their groES gene sequence lengths were 285 bp. The groESL sequences in clinical isolates of three serotypes of S. mutans were analysed for intraspecies polymorphism. The results showed that the groESL sequences could provide information for differentiation among species, but were unable to distinguish serotypes of the same species. Based on the determined sequences, a PCR assay was developed that could differentiate members of the mutans streptococci by amplicon size and provide an alternative way for distinguishing mutans streptococci from other viridans streptococci.

The aim of the current study was to assess the reliability of two enzyme immunoassays in detecting the Helicobacter pylori status of stool specimens of Turkish dyspeptic patients in the post-treatment period. Forty-eight patients with non-ulcer dyspepsia who were positive for H. pylori underwent a 1 week regimen of triple therapy. Stool samples of patients were obtained 2 and 6 weeks after eradication therapy and a [13C]urea breath test was performed 6 weeks after therapy in order to assess the reliability of mAb-based (Amplified IDEIA HpStAR, DakoCytomation) and polyclonal-antiserum-based (Premier Platinum HpSA, Meridian Diagnostics) stool antigen test kits and to compare their diagnostic accuracies. Using a minimum cutoff OD450 value of 0.19 for Amplified IDEIA HpStAR and 0.16 for Premier Platinum HpSA the sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of the tests were determined 2 and 6 weeks after completion of eradication therapy. At both the second and the sixth week in the post-treatment period the diagnostic accuracy of Amplified IDEIA HpStAR was significantly better than the Premier Platinum's (75 % versus 50 %, S2 = 6.4; P = 0.011, and 90 % versus 69 %, S2 = 6.316; P = 0.012, respectively). In light of these findings the mAb-based Amplified IDEIA HpStAR has a high diagnostic accuracy for H. pylori infection in Turkish dyspeptic patients 6 weeks after completion of eradication therapy.

Type 1 diabetes (T1D) is an autoimmune disease linked with genetic factors as well as with environmental triggers, such as virus infections, but the aetiology is still unclear. The authors analysed serum from autoantibody-positive (n = 50) and autoantibody-negative (n = 50) schoolchildren as well as children newly diagnosed with T1D (n = 47; time from diagnosis, median 5 days, interquartile range 1–12 days) for the presence and frequency of enterovirus (EV) and adenovirus sequences. The autoantibody-positive and -negative groups were part of the Karlsburg Type 1 Diabetes Risk Study of a Normal Schoolchild Population, which represents a general population without T1D first-degree relatives. There was no significant seasonality of sampling in any of the three groups investigated. EV RNA sequences were detected in 10 of 50 (20 %) autoantibody-positive children and in 17 of 47 (36 %) children newly diagnosed with T1D, but only in two of 50 (4 %) of the age- and sex-matched controls (P < 0.05, P < 0.001). Characterization of the EV amplicons by direct sequencing revealed high homology with coxsackievirus B group. For adenovirus we found no data to support an association with T1D. The data support the hypothesis that different enteroviruses may be aetiologically important as a trigger and/or accelerating factor in the process of T1D development.

Six Klebsiella pneumoniae isolates that exhibited resistance to a wide spectrum of antibiotics were recovered from the intensive care units in the First Affiliated Hospital, Zhejiang University, Hangzhou, China. All isolates contained two plasmids of approximately 95 kb and 200 kb. The 95 kb plasmid was shown to be transferable by conjugation experiments. Isoelectric focusing patterns of the β-lactamases extracted from the six transconjugants were identical, displaying five pI bands: 5.4, 7.75, 8.0, 8.2 and 8.4. The band corresponding to a pI of 7.75 could be inhibited by cloxacillin but not clavulanic acid, while the other bands could be inhibited by clavulanic acid but not cloxacillin. The 95 kb plasmid was digested with HindIII and a recombinant plasmid pT948 was obtained. The insert was found to contain bla DHA−1, regulatory gene ampR and an insertion element (IS26), which was downstream of bla DHA−1. PCR and DNA sequencing results confirmed that the 95 kb plasmid encoded at least four β-lactamase genes: bla TEM−1, bla SHV−12, bla CTX−M−3 and bla DHA−1. Epidemiological typing by PFGE of the six clinical isolates of K. pneumoniae demonstrated identical genotypic patterns. In conclusion, all results indicated that the six multi-drug resistant clinical isolates of K. pneumoniae most probably originated from one clone and caused a localized epidemic in the intensive care units.

Vancomycin-resistant enterococci (VRE) are formidable organisms renowned for their ability to cause infections with limited treatment options and their potential for transferring resistance genes to other Gram-positive bacteria. Usually associated with nosocomial infections, VRE are rarely reported as a cause of community-acquired infection. Presented here is a case of community-acquired infection due to vancomycin-resistant Enterococcus faecium. The patient had been applying herbal leaves topically to his cheek to treat a buccal space abscess, resulting in a burn of the overlying skin. From pus aspirated via the skin a pure culture of E. faecium was grown that was resistant to vancomycin with a MIC of >256 μg ml−1 by the E test and resistant to teicoplanin by disc diffusion, consistent with the VanA phenotype. The organism was suspected of contaminating the leaf and infecting the patient via the burnt skin. This case highlights the need for further studies on the community prevalence of VRE among humans and animals to define unrecognized silent reservoirs for VRE, which may pose a threat to public health.