Mycobacteria distinct from Mycobacterium avium subsp. paratuberculosis isolated from the faeces of ruminants possess IS 900 -like sequences detectable by IS 900 polymerase chain reaction: implications for diagnosis

PCR targeting the 5′ end of IS 900 has been considered specific for identification ofMycobacterium avium subsp. paratuberculosis and is frequently applied to confirm the presence of this organism in the diagnosis of Johne's disease. IS 900 PCR has also been applied to studies of the aetiology of Crohn's disease. Mycobacterium spp. isolated from the faeces of 3 clinically normal animals in 2 Australian states appeared not to be M. paratuberculosis but were positive by IS 900 PCR. The isolates were characterized using mycobactin dependency, biochemical tests, IS 900 and 16 S rRNA sequencing and restriction fragment length polymorphism (RFLP) using IS 900 as probe. DNA sequencing confirmed that the isolates had between 71% and 79% homology with M. paratuberculosis in the region of IS 900 amplified, were most closely related to Mycobacterium scrofulaceum, and confirmed the usefulness of restriction enzyme analysis of amplified product to identify the false positive results. RFLP analysis with Bst Ell detected three to five copies of the IS 900 -like element in the isolates. These were located in molecular weight fragments that were clearly different to IS 900 in previously characterized strains of M. paratuberculosis. It is likely that these isolates are environmental mycobacteria. Southern blotting with an internal probe is unlikely to provide differentiation of M. paratuberculosis from these organisms. We recommend the adoption of restriction endonuclease analysis of IS 900 PCR product as a routine precaution to prevent the reporting of false positive IS900 PCR results.     

Polymorphisms in IS1311, an insertion sequence common toMycobacterium aviumandM. aviumsubsp.paratuberculosis, can be used to distinguish between and within these species

IS1311is an insertion sequence fromMycobacterium aviumandM. aviumsubsp.paratuberculosis.Using a 180 bp fragment of IS1311as a probe, 7–10 copies of IS1311were revealed in strains ofM. aviumsubsp.paratuberculosis.With a given restriction enzyme, the restriction fragment length polymorphism patterns obtained from isolates ofM. aviumsubsp.paratuberculosisfrom cattle were all identical, but they differed from those of isolates from sheep, which could be separated into two types. A 1259 bp fragment of IS1311produced by polymerase chain reaction (PCR) from two isolates ofM. aviumsubsp.paratuberculosisfrom cattle and two isolates from sheep was sequenced and compared to the sequence known fromM. avium.Apart from five point differences the sequences were identical. Restriction endonuclease analysis (REA) of the PCR product was used to distinguish isolates ofM. aviumsubsp.paratuberculosisfromM. avium, in addition to the conventional test for IS900.In isolates ofM. aviumsubsp.paratuberculosisfrom cattle the IS1311gene was polymorphic at position 223, which enabled isolates from sheep and cattle to be distinguished by PCR-REA. These simple tests will be used to enhance disease control programmes for Johne's disease in ruminants. The findings of this study raise interesting questions about the evolution ofM. aviumsubsp.paratuberculosis.     

Detection and characterization of thefimAgene ofEscherichia coliO157:H7

An expected 850-bp DNA fragment containingfimA, the structural gene for type 1 fimbriae, and flanking sequences was amplified from 39 (of 46) pathogenic and commensal strains ofEscherichia coliusing the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) analysis of the amplified products showed 13HinPI and fourSau96I restriction profiles among these 39E. colistrains, revealing the polymorphic nature of this allele. A unique RFLP pattern was shared byE. coliO157:H7, O157:H^- and a few O55 serotype strains. DNA sequence analysis of thefimAregion demonstrated thatE. coliO157:H7 strain 933 and O157:H⁻strain E32511 contained identical DNA sequences that were distinct from otherE. colistrains, especially a 16-bp sequence 5′ tofimAthat was conspicuously absent only inE. coliO157 strains. Exploiting these differences, a PCR assay was developed that amplifies a 936-bp fragment from allE. coliO157:H7 strains examined to date. This PCR assay offers a simple, rapid, and reliable means to detectE. colistrains of the O157:H7 serotype.     

Specific detection and quantification of the phytopathogenic agent ‘Candidatus Phytoplasma prunorum’

‘Candidatus Phytoplasma prunorum’ is a wall-less bacterium associated with European stone fruit yellows (ESFY), a severe disease of Prunus spp. (mainly apricot and Japanese plum trees). It can be spread by one insect vector, Cacopsylla pruni, and by the trade of infected material. The availability of PCR-based methods allowing a sensitive and specific detection of ‘Ca. P. prunorum’ is crucial for this phytoplasma because, at present, it is uncultured and cannot be detected serologically. We developed a PCR test which, in contrast to the existing detection tools, provides a fast, specific and sensitive detection of ‘Ca. P. prunorum’ in plants and insects. For studies requiring an absolute quantification of the phytoplasma titer, the same primers were used to develop a real-time PCR assay, including a standard for C. pruni. The sensitivity of these molecular tools was compared by serial dilutions and their specificity was assessed both in silico and experimentally for reference strains and field samples of the closely related phytoplasma ‘Ca. P. prunorum’, ‘Ca. P. pyri’ (pear decline agent) and ‘Ca. P. mali’ (apple proliferation agent), as well as for representative strains of the ‘Ca. Phytoplasma’ genus.     

A PCR-RFLP assay for identification of Anisakis simplex from different geographical regions

Anisakis simplex, a nematode from the family Anisakidae, is a parasite of fish and mammals. It is a casual agent of a human disease called anisakiosis. We found that the assay based on PCR amplification of the ITS-1–5·8 S–ITS-2 fragment of rDNA and subsequent restriction fragment length polymorphism, previously described on the basis of A. simplex isolated solely from one geographical region, can be used as a general test for identification of this worm species. The restriction patterns analysed for four restriction enzymes were found to be identical in the case of allA. simplex individuals isolated from as different geographical regions as Baltic Sea, Norwegian Sea, Bering Sea and Sea of Okchotsk. Moreover, our results support the previously proposed hypothesis, based on the studies of isoenzymes, that there is a remarkable genetic homogeneity within A. simplex from different geographical regions.     

Amplification of the DNA polymerase I gene of Treponema pallidum from whole blood of persons with syphilis

Previous reports suggest that Treponema pallidum bacteremia occurs in persons with syphilis exposure (‘incubating syphilis’) and in persons with primary or secondary syphilis. During a recent syphilis outbreak, whole blood samples from 32 persons with suspected syphilis or syphilis exposure were screened using polymerase chain reaction (PCR) to amplify the DNA polymerase I gene (polA) of T. pallidum. Of the 32 samples, polA was amplified from 13 (41%). Of these 13, three were determined to have incubating syphilis; two had primary or secondary syphilis and eight had latent syphilis. This study demonstrates that spirochetemia can occur throughout the course of T. pallidum infection.     

Multiplex PCR for detection of trait and virulence factors in enterohemorrhagic Escherichia coli serotypes

A multiplex PCR assay was developed which allowed the simultaneous detection of five trait genes or virulence markers in enterohemorrhagic Escherichia coli (EHEC) serotypes. A primer pair, designed to detect a single base-pair mutation in the uidA gene, is specific only for the prototypic EHEC of O157:H7 serotype and its toxigenic, non-motile variants. In a similar way, primers to the eaeA gene of the γ-intimin derivative specifically detects strains in the EHEC 1 clonal group, which consists mostly of O157:H7 and some O55:H7 serotypes. The other three primer pairs, specific for stx1, stx2 and both variants of ehxA genes, will detect the presence of these virulence genes in all EHEC serotypes. Analysis of 34 strains, including various serotypes of EHEC, Shiga toxin-producing E. coli and enteropathogenic E. coli, confirmed that the multiplex PCR assay detected the presence of these genes in a manner consistent with the known genotype of each respective strains.     

Application of PCR for <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis> detection in children with community-acquired pneumonia

Between April 2002 and March 2003, to detect Mycoplasma pneumoniae by polymerase chain reaction (PCR), a primer set designed for the 16S rRNA gene was used to examine clinical samples from 369 children with community-acquired pneumonia. Samples were collected from 12 Japanese institutions participating in a study group concerning acute respiratory infectious diseases. The sensitivity of primers – 2 CFU per reaction tube, using M. pneumoniae M129, a standard strain – was calculated to represent 1.1 × 10³ M. pneumoniae organisms adherent to the tip of the swab used to collect clinical samples. Results for PCR were obtained within 2.6h. Cases identified by PCR, cultures, and serologic tests were 68 (18.4%), 53 (14.4%), and 76 (20.6%) respectively. Among 57 PCR-positive patients tested serologically, 56 showed a significant elevation or rise in antibody titer. PCR positivity was high among patients prescribed -lactam antibiotics (86.7%) or no antibiotic (87.0%) before PCR analysis, but was low among patients receiving macrolides, new quinolones, or tetracyclines (37.5%). We concluded that the PCR constructed by us had a high probability for confirming a diagnosis of M. pneumoniae pneumonia and for guiding antibiotic choice for patients not yet treated.     

Molecular detection and quantification of the protozoan Bonamia ostreae in the flat oyster, Ostrea edulis

Bonamia ostreae is an intracellular protozoan which is recognized as a cause of mortality in European populations of flat oysters (Ostrea edulis). Based on the recent characterization of actin genes of B. ostreae, specific primers were designed for real-time PCR using SYBR^® Green chemistry. Specificity was demonstrated by the unique melting temperature peak observed in positive samples and by the lack of amplification in samples of oysters infected by closely related parasites, including Bonamia exitiosa. A calibration curve using a cloned template was defined to estimate copy number. The assay had a 6 log- dynamic range, mean inter- and intra-assay variation coefficients of <1% and a minimum detection limit of 50 gene copies per reaction. Using infected oyster samples as templates, the assay was at least 10-fold more sensitive than conventional PCR. The quantitative assay was applied to test 132 oysters, and results were compared with the heart imprint method. There was a strong correlation between both techniques, and the results showed that the real-time PCR assay should be useful for studies of the ecology of B. ostreae and its host–parasite relationship.     

Controlled multiplex PCR of enterotoxigenicClostridium perfringensstrains in food samples

A controlled multiplex polymerase chain reaction (PCR) for the detection ofClostridium(C.)perfringensenterotoxin gene (cpe) was established and compared with anin vitroantigenic detection method. ThecpePCR and the classical method of electric immunodiffusion gave identical results. The predicted specific amplicon of thecpegene was generated from all of the tested enterotoxigenicC. perfringensstrains. In contrast, cultures of any otherClostridiumspecies tested by PCR were negative (100% sensitivity, 100% specificity). Addition of an alphatoxin (plc) gene specific PCR as an in process control reaction was performed in order to prevent false negative PCR results. The PCR detection limit was 0·5 ng of genomicC. perfringensDNA per ml of bouillon culture. By contaminating minced meat withC. perfringensreference strains, the multiplex PCR was established as a tool for routine diagnostic laboratories. The detection limit was approximately 3·0×10⁵C. perfringenscells per gram meat. The results demonstrate the multiplex PCR as an easy, specific, sensitive and time saving diagnostic procedure. Application of this improved method should enhance the knowledge concerning epidemiological aspects of food borne diseases caused by enterotoxigenicC. perfringens.     

Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction

Polymerase chain reaction (PCR) protocols were established for specific detection of the tdh and trh genes, the virulence marker genes of Vibrio parahaemolyticus encoding two related hemolysins. The tdh and trh genes are known to have sequence divergence of up to 3 · 3% and 16%, respectively. Attempts were made to find suitable primer pairs and annealing temperatures to detect each gene without fail. DNAs extracted from 36 representative strains of V. parahaemolyticus were used in the initial screening with various combinations of primer pairs and annealing temperatures. The combinations of primer pairs and annealing temperatures selected were then tested with DNAs extracted from 227 more strains of V. parahaemolyticus and from 133 bacterial strains belonging to 40 species other than V. parahaemolyticus. PCR protocols (primer pairs and annealing temperatures) were established that gave identical results to those obtained with the tdh- and trh-specific polynucleotide probes. These protocols established for the tdh and trh genes could detect 400 fg (100 cells) of cellular DNA carrying the respective gene. Spike experiments demonstrated that the sensitivities of the established PCRs were reduced by a factor of 10⁴–10⁵ by an inhibitor(s) present in a normal faecal sample, indicating the need for either DNA extraction or enrichment of the faecal sample in alkaline peptone water for 4 h before the PCR of faecal samples.     

Polymerase chain reaction for the detection ofPseudomonas aeruginosa,Stenotrophomonas maltophiliaandBurkholderia cepaciain sputum of patients with cystic fibrosis

Occurrence ofPseudomonas aeruginosa,Stenotrophomonas (Xanthomonas) maltophiliaandBurkholderia (Pseudomonas) cepaciain sputum of cystic fibrosis (CF) patients was demonstrated with a simple and rapid polymerase chain reaction (PCR) technique. The PCR was performed with a set of three primer pairs based on 16S rRNA sequences after sputum preparation with dithiothreitol and NaOH lysis. All three pathogens could be individually detected by the use of this technique. To prevent carry-over contamination, dUTP and uracil-N-glycosylase were included in the reaction. The amplicons were visualized by agarose gel electrophoresis. Sputum culture was performed on all samples. Ninety specimens from CF patients were analysed. The sensitivity for the detection ofP. aeruginosawas 37/40 (93%) compared to culture. Bacterial growth ofP. aeruginosawas found in three cases, where PCR amplicons were not detected, while PCR was positive in five cases, where culture did not reveal the presence of this bacterium. For this reason, the specificity was 45/50 (90%). ForS. maltophilia, the PCR was less sensitive than culture (positive in three of six cases). In our series,B. cepaciawas detected by culture in one case and this was also detected by PCR. There were no false-positive PCR results regardingS. maltophiliaorB. cepacia. Thus, combined PCR-based detection of these three clinically relevant bacteria in sputum samples from CF patients can be performed by a reliable technique in a relatively simple manner. The present data indicate a high sensitivity and specificity forP. aeruginosa. The lower sensitivity observed for the detection ofS. maltophiliain sputum andB. cepacia, as estimated from laboratory strains, may depend on PCR conditions and genetic heterogeneity, respectively. The greatest gains with this method can be made when it is used for the early detection ofP. aeruginosain sputum-producing CF patients.     

Novel NPR1 polymorphic variants and its exclusion as a candidate gene for medullary cystic kidney disease (ADMCKD) type 1

Autosomal dominant medullary cystic kidney disease (ADMCKD) is an adult-onset heterogeneous genetic nephropathy characterized by salt wasting and end-stage renal failure. The gene responsible for ADMCKD-1 was mapped on chromosome 1q21 and it is flanked proximally by marker D1S498 and distally by D1S2125, encompassing a region of 8 c . Within this region there are a large number of transcribed genes including NPR1 that encodes the atrial natriuretic peptide receptor 1. This receptor plays a crucial role in regulation of blood pressure by facilitating salt excretion. Based on its function we hypothesized this gene as a reasonable candidate for the MCKD1 locus. DNA mutation screening was performed on the entire NPR1 gene-coding sequence and some of the 5′-UTR and 3′-UTR sequences. The samples investigated belonged to patients of five large ADMCKD-1 Cypriot families. The screening revealed two novel polymorphisms, one intragenic at amino acid position 939, which was occupied by either arginine or glutamine, and a second one located in the 3′-UTR, 29 nucleotides downstream of the NPR1 stop codon. The latter was a single nucleotide C insertion/deletion in a stretch of three or four Cs. No relationship was present between any allele of the two polymorphisms and the disease, as both alleles were observed in both affected and healthy subjects. In addition, no association was observed between the disease and another rare 8-bp deletion polymorphism at the 5′-UTR of NPR1 and the disease. Based on these findings it is unlikely thatNPR1 is the same as the MCKD1 gene, although it is presently unknown whether it plays a disease modifying role.     

No regional spread of vancomycin-resistant enterococci with <Emphasis Type="Italic">vanA</Emphasis> or <Emphasis Type="Italic">vanB</Emphasis> in Kitakyushu,Japan

Outbreaks of vancomycin-resistant enterococci (VRE) infection occur sporadically in Japan, and their frequency has been gradually increasing. We experienced a nosocomial outbreak of VRE in two hospitals in the city of Kitakyushu, and the spread of VRE strains was suspected in this area. To examine the prevalence rate of infection and colonization of VRE in Kitakyushu, we screened a total of 24297 clinical samples from patients in hospitals and clinics in Kitakyushu from October through December 2002 for VRE. The isolates screened as positive for VRE accounted for 2.3% (566/24297) of the tested clinical samples. Polymerase chain reaction (PCR) analyses for vanA, vanB, vanC1, and vanC2/3 were performed to confirm the screening test results. Neither vanA nor vanB genes were detected in any isolates. The 265 vanC1-positive isolates were Enterococcus gallinarum, and the 150 vanC2/3-positive isolates were E. casseliflavus. Other Enterococcus species were negative in this PCR-detection test. In this study, the PCR procedure was considered reliable and successful because although neither vanA nor vanB was detected, vanC1 and vanC2/3 were completely detectable. Therefore, we concluded that the regional spread of VRE with vanA and vanB had not occurred in Kitakyushu in 2002. In the near future, the prevalence of VRE with vanA or vanB is likely to increase in Japan, as it has in other countries. We should continue to find and prevent nosocomial outbreaks of infection and colonization by VRE.     

Emergence of multiresistant variants of the community-acquired methicillin-resistant Staphylococcus aureus lineage ST1-SCCmecIV in 2 hospitals in Rio de Janeiro, Brazil

Usually, community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is susceptible to a variety of non–β-lactam drugs. These isolates commonly display SCCmecIV and are associated with community-acquired infections. More recently, CA-MRSA has been isolated from health-care–associated diseases. We characterized MRSA isolates from 2 hospitals in Rio de Janeiro area to assess the entry of new lineages. The isolates were primary genotyped using a combination of molecular typing methods including SCCmec, restriction modification test, and Panton–Valentine leukocidin (PVL) detection. Pulsed-field gel electrophoresis was carried out for representatives of each lineages found. Disk diffusion test was performed as recommended by the Clinical and Laboratory Standards Institute. SCCmecIV was the predominant cassette mec detected. The most frequent MRSA lineage, a PVL nonproducer, was allocated in the CC1-SCCmecIV. It was found that 56% of these isolates were resistant to 3 or more non–β-lactam drugs. Multilocus sequence typing of a representative of the CC1 isolates supported our finds that multiresistant variants of a CA-MRSA lineage (ST1-SCCmecIV) emerged in this city.     

In vitro induction of resistance to metronidazole, and analysis of mutations in rdxA and frxA genes from Helicobacter pylori isolates

In clinical Helicobacter pylori isolates, metronidazole resistance has been associated with mutations in the rdxA and frxA genes. The aim of this study was to examine the role of the rdxA and frxA genes after the in vitro induction of metronidazole resistance. A total of five suscep-tible H. pylori isolates were initially exposed to different subinhibitory metronidazole concentrations to induce in vitro resistance to metronidazole. Susceptible and resistant strains after the in vitro induction of resistance were examined to evaluate mutations of the rdxA and frxA genes by sequence analysis. After the in vitro induction of resistance, analysis revealed that two and four susceptible strains developed resistance when cultured with 0.3µg/ml and 0.6µg/ml of metronidazole, respectively. Before and after the induction of resistance, none of the susceptible strains that developed low and moderate levels of resistance presented any mutation in either of the evaluated genes, whereas strains with high-level metronidazole resistance contained a simple mutation of the frxA gene, but no specific changes in the rdxA gene. Strains with moderate-level resistance contained both single and multiple mutations of rdxA and frxA, respectively, and the low-level-metronidazole-resistant strain contained a single mutation in the frxA gene, without any significant change in the rdxA gene. In this study, the strains that developed resistance were mainly associated with mutations of the frxA gene, suggesting the possibility that inactivation of this gene could originate metronidazole resistance. The results after the in vitro induction of resistance to metronidazole suggested the presence of additional metronidazole resistance mechanisms, other than mutations of the rdxA and/or frxA genes.     

Identification of penicillin and other beta-lactam resistance inStreptococcus pneumoniae by polymerase chain reaction

To identify penicillin (Pc) and other β-lactam resistance in 310 clinical isolates ofStreptococcus pneumoniae by polymerase chain reaction (PCR), 3 sets of primers were designed to amplify Pc-binding protein (PBP) genes previously detected in Pc-susceptible strains: 1) a 430-bp fragment of thepbp1a gene, 2) a 292-bp fragment of thepbp2x gene, and 3) a 77-bp fragment of thepbp2b gene. The amplified regions of each PBP gene were positioned in highly divergent sequences of Pc-resistantS. pneumoniae. In other words, isolates for which these DNA fragments were detected were regarded as possessing sequences almost the same as that of the susceptible R6 strain and those for which these DNA fragments were not detected were assumed to have mutations. A set of primers that amplify 273 bp of the autolysin (lytA) gene to identifyS. pneumoniae was applied as well. Of 166 isolates for which the minimum inhibitory concentration (MIC) of Pc were ≤0.06 μg/mL, 83 (50.0%) were confirmed to be true susceptible strains with no PBP gene mutation and most of the remaining strains were found to possesspbp2x mutation. In contrast, most of 109 isolates for which the MIC of Pc were ≥0.5 μg/mL were confirmed to possess mutations in all three PBP genes. Thirty-five strain for which the MIC of Pc ranged from 0.125 to 0.25 μg/mL possessed various PBP gene mutations. The relationships between susceptibilities to 9 β-lactams ofS. pneumoniae and PBP gene mutation were analyzed by multiple regression analysis. Antibiotics were classified into 4 types according to the differences in PBP gene mutation affecting their MIC levels, 1) the MIC of Pc and ampicillin were affected bypbp1a andpbp2b mutations; 2) those of cefotaxime, cefpodoxime, and cefditoren were affected clearly bypbp2x mutation; 3) those of cefaclor and cefdinir were affected more strongly bypbp1a mutation than thepbp2x; and 4) the MIC of faropenem and imipenem were affected strongly bypbp2b mutation. These findings suggest that it may be possible to easily determine whether aS. pneumoniae isolate is susceptible or resistant to Pc, cefotaxime, and other β-lactams by applying PCR using a combination of primers.     

Dissemination of extended-spectrum β-lactamase-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> in Thai hospitals

Fifty clinical isolates of Klebsiella pneumoniae and Escherichia coli with reduced susceptibility to third-generation cephalosporins, collected from 11 hospitals in Thailand, were studied. All isolates were found to produce extended-spectrum β-lactamase (ESBL), as judged by double-disk synergy and combination disk methods. Most ESBL-producing K. pneumoniae isolates were resistant to ceftazidime (94%) and aztreonam (90%). In contrast, most ESBL-producing E. coli isolates were resistant to ceftriaxone (95%) and cefotaxime (74%). Plasmid DNA was isolated and β-lactamase genes were identified by PCR and sequencing. We found that SHV-12 and CTX-M-14 were the main ESBLs responsible for resistance in K. pneumoniae and E. coli, respectively. SHV-27, SHV-28, and CTX-M-14 were detected in three, two, and four K. pneumoniae isolates, respectively. A high genetic diversity among ESBL-producing K. pneumoniae and E. coli isolates was observed. In addition, the finding of a few isolates that produced identical restriction patterns on pulsed field gel electrophoresis (PFGE) suggests the clonal spread of resistant bacteria within the hospital.     

Alarming trend of clarithromycin-resistant Streptococcus pyogenes in Japan (1998–2002)

We investigated annual changes in clarithromycin resistance and resistance genes in 579 strains of Streptococcus pyogenes isolated from patients with symptomatic respiratory tract infections who visited primary medical institutions during the 5-year period from 1998 to 2002. The minimum inhibitory concentrations (MICs) of clarithromycin for S. pyogenes were measured using the standard broth microdilution method according to the National Committee for Clinical Laboratory Standards (NCCLS) guidelines, and strains showing MICs of 1µg/ml or greater were regarded as being resistant to clarithromycin, according to the resistance standard specified by the NCCLS. The rates of S. pyogenes resistance to clarithromycin were 7.3% overall, 5.8% in 1998, 4.9% in 1999, 7.7% in 2000, 6.4% in 2001, and 11.1% in 2002. While the annual rates fluctuated slightly each year, an overall tendency to increase was observed during the 5-year period. Regarding the macrolide-resistance genes in the macrolide-resistant strains, mefA/E (+)/ ermB(–) was the most common genotype detected in these strains, while the ermB (+)/ mefA/E (–) and mef A/E (–)/ ermB (–) genotypes were detected at about the same rate. The MICs of clarithromycin for the ermB (+) strains tended to be higher than those of the mefA/E (+) strains, but some mefA/E (–) / ermB (–) strains also exhibited high MICs of clarithromycin, similar to those of the ermB (+) strains. The above results indicate that the number of clarithromycin-resistant strains of S. pyogenes is gradually increasing and that the resistance is becoming stronger; thus, special attention must be paid to the appearance of macrolide-resistant strains of S. pyogenes.     

Rapid identification of noncapsulated Streptococcus pneumoniae in nasopharyngeal samples allowing detection of co-colonization and reevaluation of prevalence

Noncapsulated pneumococci are atypical Streptococcus pneumoniae that lack a capsule and therefore do not react with any available antisera. These isolates, which are often referred as nontypeable pneumococci (NTPn), are difficult to identify as their differentiation from closely related species such as Streptococcus pseudopneumoniae and other streptococcus of the mitis group is not always straightforward. We developed a low-cost and easy assay to detect and quantify NTPn in primary samples (which may contain multiple species) obtained from nasopharyngeal swabs. The strategy is based on a multiplex polymerase chain reaction targeting lytA, cpsA, aliB-like ORF2, and 16S rDNA genes, plus a restriction fragment length polymorphism assay to differentiate typical from atypical lytA. The application of the proposed methodology to over 500 nasopharyngeal samples found that the prevalence of NTPn in colonization was 3-fold higher than that estimated by routine methods (from 2.9% to 8.6% in the study collection). The international clone Norway^NTST344 was the major clone identified.