Somay Yamagata Murayama

DETECTION OF A WIDE RANGE OF MEDICALLY IMPORTANT FUNGI BY THE POLYMERASE CHAIN REACTION

A polymerase chain reaction (PCR) method was developed that was capable of detecting a wide range of medically important fungi from clinical specimens. The primer pair was designed in conserved sequences of 18S-ribosomal RNA genes shared by most fungi. The lower limit of detection of this PCR technique was 1 pg of Candida albicans genomic DNA by ethidium bromide staining and 100 fg after Southern analysis. A 687-bp product was amplified successfully by PCR from all 78 strains of 25 medically important fungal species studies, including Candida spp., Hansenula spp., Saccharomyces cerevisiae, Cryptococcus neoformans, Trichosporon beigelii, Malassezia furfur, Pneumocystis carinii, Aspergillus spp., and Penicillium spp., but not from any strains of Mucor spp., Escherichia coli, or methicillin-resistant Staphylococcus aureus (MRSA), calf thymus or human placenta. This specificity was subsequently confirmed by Southern analysis. PCR analysis of blood specimens collected from mice systemically infected with C. albicans and clinical samples including blood, cerebrospinal fluid and sputum appeared to be a more sensitive diagnostic method for invasive fungal infections than a conventional blood culture technique.

Rapidly Increasing Prevalence of β-Lactamase-Nonproducing, Ampicillin-Resistant Haemophilus influenzae Type b in Patients with Meningitis

ABSTRACT A total of 395 Haemophilus influenzae strains from 226 Japanese institutions participating in the Nationwide Surveillance Study Group for Bacterial Meningitis were received from 1999 to 2002. All strains were analyzed by PCR to identify the resistance genes, and their susceptibilities to β-lactam agents were determined. Of these strains, 29.1% were β-lactamase nonproducing and ampicillin (AMP) susceptible (BLNAS) and lacked all resistance genes; 15.4% were β-lactamase producing and AMP resistant and had the blaTEM-1 gene; 30.6% were β-lactamase nonproducing and AMP resistant (low-BLNAR) and had a Lys-526 or His-517 amino acid substitution in ftsI encoding PBP 3; 13.9% were β-lactamase nonproducing and AMP resistant (BLNAR) and had an additional substitution of Thr-385 in ftsI; 9.1% were amoxicillin-clavulanic acid resistant (BLPACR I) and had the blaTEM-1 gene and a Lys-526 or His-517 amino acid substitution in ftsI; and 1.8% showed resistance similar to that of the BLPACR I group (BLPACR II) but had blaTEM-1 gene and ftsI substitutions, as was the case for the BLNAR strains. All but three strains were serotype b. The prevalence of BLNAR strains has increased rapidly: 0% in 1999, 5.8% in 2000, 14.1% in 2001, and 21.3% in 2002. The MICs at which 90% of BLNAR isolates were inhibited were as follows: AMP, 16 μg/ml; cefotaxime, 1 μg/ml; ceftriaxone, 0.25 μg/ml; and meropenem, 0.5 μg/ml. All of these values were higher than those for the BLNAS counterpart strains. The relatively wide distributions of the β-lactam MICs for BLNAR strains presumably reflect variations in ftsI gene mutations. Pulsed-field gel electrophoresis suggested the rapid spread of specific H. influenzae type b strains throughout Japan. Expedited vaccination, rapid identification, and judicious antibiotic use could slow their spread.

Comprehensive detection of causative pathogens using real-time PCR to diagnose pediatric community-acquired pneumonia

We have developed a real-time reverse transcription-PCR (RT-PCR) method to detect 13 respiratory viruses: influenza virus A and B; respiratory syncytial virus (RSV) subgroup A and B; parainfluenza virus (PIV) 1, 2, and 3; adenovirus; rhinovirus (RV); enterovirus; coronavirus (OC43); human metapneumovirus (hMPV); and human bocavirus (HBoV). The new method for detection of these viruses was applied simultaneously with real-time PCR for the detection of six bacterial pathogens in clinical samples from 1700 pediatric patients with community-acquired pneumonia (CAP). Of all the patients, 32.5% were suspected to have single bacterial infections; 1.9%, multiple bacterial infections; 15.2%, coinfections of bacteria and viruses; 25.8%, single viral infections; and 2.1%, multiple viral infections. In the remaining 22.6%, the etiology was unknown. The breakdown of suspected causative pathogens was as follows: 24.4% were Streptococcus pneumoniae, 14.8% were Mycoplasma pneumoniae, 11.3% were Haemophilus influenzae, and 1.4% were Chlamydophila pneumoniae. The breakdown of viruses was as follows: 14.5% were RV, 9.4% were RSV, 7.4% were hMPV, 7.2% were PIV, and 2.9% were HBoV. The new method will contribute to advances in the accuracy of diagnosis and should also result in the appropriate use of antimicrobials.

Complete genome sequencing and analysis of a Lancefield group G Streptococcus dysgalactiae subsp. equisimilis strain causing streptococcal toxic shock syndrome (STSS)

BackgroundStreptococcus dysgalactiae subsp. equisimilis (SDSE) causes invasive streptococcal infections, including streptococcal toxic shock syndrome (STSS), as does Lancefield group A Streptococcus pyogenes (GAS). We sequenced the entire genome of SDSE strain GGS_124 isolated from a patient with STSS.ResultsWe found that GGS_124 consisted of a circular genome of 2,106,340 bp. Comparative analyses among bacterial genomes indicated that GGS_124 was most closely related to GAS. GGS_124 and GAS, but not other streptococci, shared a number of virulence factor genes, including genes encoding streptolysin O, NADase, and streptokinase A, distantly related to SIC (DRS), suggesting the importance of these factors in the development of invasive disease. GGS_124 contained 3 prophages, with one containing a virulence factor gene for streptodornase. All 3 prophages were significantly similar to GAS prophages that carry virulence factor genes, indicating that these prophages had transferred these genes between pathogens. SDSE was found to contain a gene encoding a superantigen, streptococcal exotoxin type G, but lacked several genes present in GAS that encode virulence factors, such as other superantigens, cysteine protease speB, and hyaluronan synthase operon hasABC. Similar to GGS_124, the SDSE strains contained larger numbers of clustered, regularly interspaced, short palindromic repeats (CRISPR) spacers than did GAS, suggesting that horizontal gene transfer via streptococcal phages between SDSE and GAS is somewhat restricted, although they share phage species.ConclusionGenome wide comparisons of SDSE with GAS indicate that SDSE is closely and quantitatively related to GAS. SDSE, however, lacks several virulence factors of GAS, including superantigens, SPE-B and the hasABC operon. CRISPR spacers may limit the horizontal transfer of phage encoded GAS virulence genes into SDSE. These findings may provide clues for dissecting the pathological roles of the virulence factors in SDSE and GAS that cause STSS.

Rapid detection of eight causative pathogens for the diagnosis of bacterial meningitis by real-time PCR

We aimed to detect causative pathogens in cerebrospinal fluid (CSF) collected from patients diagnosed with bacterial meningitis by real-time polymerase chain reaction (PCR). In addition to Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae described previously, five other pathogens, Neisseria meningitidis, Escherichia coli, Streptococcus agalactiae, Staphylococcus aureus, and Listeria monocytogenes, were targeted, based on a large-scale surveillance in Japan. Results in CSF from neonates and children (n = 150), and from adults (n = 18) analyzed by real-time PCR with molecular beacon probes were compared with those of conventional culturing. The total time from DNA extraction from CSF to PCR analysis was 1.5 h. The limit of detection for these pathogens ranged from 5 copies to 28 copies per tube. Nonspecific positive reactions were not recognized for 37 microorganisms in clinical isolates as a negative control. The pathogens were detected in 72.0% of the samples by real-time PCR, but in only 48.2% by culture, although the microorganisms were completely concordant. With the real-time PCR, the detection rate of H. influenzae from CSF was high, at 45.2%, followed by S. pneumoniae (21.4%), S. agalactiae (2.4%), E. coli (1.8%), L. monocytogenes (0.6%), and M. pneumoniae (0.6%). The detection rate with PCR was significantly better than that with cultures in patients with antibiotic administration (χ2 = 18.3182; P = 0.0000). In conclusion, detection with real-time PCR is useful for rapidly identifying the causative pathogens of meningitis and for examining the clinical course of chemotherapy.

Capsular Type and Antibiotic Resistance in Streptococcus agalactiae Isolates from Patients, Ranging from Newborns to the Elderly, with Invasive Infections

ABSTRACT Streptococcus agalactiae isolates (n = 189) from patients with invasive infections were analyzed for capsular type by PCR, for antimicrobial susceptibility, and for the presence of resistance genes. In contrast to the predominance of capsular type III in children, types Ib and V were most common among adults. All 45 levofloxacin-resistant strains had two amino acid substitutions, Ser81Leu in the gyrA gene and Ser79Phe in the parC gene, and showed similar pulsed-field gel electrophoresis patterns.

“Candidatus Helicobacter heilmannii” from a Cynomolgus Monkey Induces Gastric Mucosa-Associated Lymphoid Tissue Lymphomas in C57BL/6 Mice

ABSTRACT Both Helicobacter pylori and “Candidatus Helicobacter heilmannii” infections are associated with peptic ulcers, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphomas. However, good animal models of H. pylori clinical diseases are rare. In this study, we aimed to establish an animal model of “Candidatus Helicobacter heilmannii” gastric MALT lymphoma. We used a urease-positive gastric mucosal and mucus homogenate from a cynomolgus monkey maintained in C57BL/6 mouse stomachs. The bacterium in the homogenate was identified as “Candidatus Helicobacter heilmannii” based on a DNA sequence analysis of the 16S rRNA and urease genes. Mucosal and mucus homogenates were used to inoculate C57BL/6 mice, which were then examined for 24 months. We observed a gradual increase in the surface area of protrusive lesions in almost all infected C57BL/6 mouse fundic stomachs 6 months after infection. Light microscopic observations revealed an accumulation of B lymphocytes along with destruction of glandular elements and the presence of lymphoepithelial lesions consistent with low-grade MALT lymphomas. Electron microscopic observation revealed numerous “Candidatus Helicobacter heilmannii” bacilli in the fundic glandular lumen, the intracellular canaliculi, and the cytoplasm of intact cells, as well as damaged parietal cells. In conclusion, “Candidatus Helicobacter heilmannii” induced gastric MALT lymphomas in almost 100% of infected C57BL/6 mice after a 6-month period associated with the destruction of parietal cells.

Distribution of emm type and antibiotic susceptibility of group A streptococci causing invasive and noninvasive disease.

To determine the prevalence of macrolide antibiotic and levofloxacin resistance in infections with Streptococcus pyogenes (group A streptococcus or GAS), strains were collected from 45 medical institutions in various parts of Japan between October 2003 and September 2006. Four hundred and eighty-two strains from patients with GAS infections were characterized genetically. Strains were classified into four groups according to the type of infection: invasive infections (n=74) including sepsis, cellulitis and toxic-shock-like syndrome; acute otitis media (AOM; n=23); abscess (n=53); and pharyngotonsillitis (n=332). Among all strains, 32 emm types were identified; emm1 was significantly more common in invasive infections (39.2 %) and AOM (43.5 %) than in abscesses (3.8 %) or pharyngotonsillitis (10.2 %). emm12 and emm4 each accounted for 23.5 % of pharyngotonsillitis cases. Susceptibility of GAS strains to eight beta-lactam agents was excellent, with MICs of 0.0005-0.063 mug ml(-1). Macrolide-resistant strains accounted for 16.2 % of all strains, while the percentages of strains possessing the resistance genes erm(A), erm(B) and mef(A) were 2.5 %, 6.2 % and 7.5 %, respectively. Although no strains with high resistance to levofloxacin were found, strains with an MIC of 2-4 mug ml(-1) (17.4 %) had amino acid substitutions at either Ser-79 or Asp-83 in ParC. These levofloxacin-intermediately resistant strains included 16 emm types, but macrolide-resistant strains were more likely than others to represent certain emm types.

Molecular emm genotyping and antibiotic susceptibility of Streptococcus dysgalactiae subsp. equisimilis isolated from invasive and non-invasive infections

To analyse the characteristics of infections caused by Streptococcus dysgalactiae subsp. equisimilis, clinical isolates (n=145) were collected at 11 medical institutions between September 2003 and October 2005. These isolates belonged to Lancefield group A (n=5), group C (n=18) or group G (n=122). Among all isolates, 42 strains were isolated from sterile samples such as blood, synovial fluid and tissue specimens from patients who were mostly over 50 years with invasive infections, and included seven cases of streptococcal toxic shock syndrome and necrotizing fasciitis. In contrast, the remaining 103 were isolated mainly from patients of all age groups with non-invasive infections such as pharyngotonsillitis. These isolates were classified into 25 types based on emm genotyping. A significant difference in emm types was observed between isolates from invasive and non-invasive infections (P<0.001): stG485, stG6792 and stG2078 predominated among isolates from invasive infections. A phylogenetic tree of complete open reading frames of emm genes in this organism showed high homology with those of Streptococcus pyogenes, but not with those of other streptococci. The presence of five different clones was estimated based on DNA profiles of isolates from invasive infections obtained by PFGE. Genes for resistance to macrolides [erm(A), three isolates; erm(B), five isolates; mef(A), seven isolates] and levofloxacin (mutations in gyrA and parC, four isolates) were identified in this organism. These results suggest the need for further nationwide surveillance of invasive infections caused by S. dysgalactiae subsp. equisimilis.

The lipolytic enzymes activities of Malassezia species

Malassezia yeasts are part of the cutaneous microflora commonly found on animals and human and may sometimes cause various opportunistic skin diseases. As most of Malassezia species show lipid-dependency, lipolytic enzymes such as lipase and phospholipase are necessary for them to obtain useful lipids from the environment. Consequently, these enzymes are thought to play an important role in the growth and pathogenicity of Malassezia. Here we analyze and compare extracellular lipase and phospholipase activities of several Malassezia species cultivated under common growth conditions. M. globosa showed the highest lipase activity of all of the Malassezia species included in our studies. The lipid-independent M. pachydermatis also showed high lipase and phospholipase activity. These results indicate that this Malassezia species are capable of utilizing lipids well in contrast to the other lipid-dependent species of the genus. Our data suggest that lipase may be a pathogenic factor in the skin disease associated with Malassezia and provide an explanation as to why M. globosa is an important pathogenic species in several human skin diseases despite its slow rate of growth.