- Volume 61, Issue 10, 2012

Multiplex PCR was used to detect genes encoding selected virulence determinants associated with strains of Escherichia coli with K1 antigen (K1+) and non-K1 E. coli (K1−). The prevalence of the fimA, fimH, sfa/foc, ibeA, iutA and hlyF genes was studied for 134 (67 K1+ and 67 K1−) E. coli strains isolated from pregnant women and neonates. The fimA gene was present in 83.6 % of E. coli K1+ and in 86.6 % of E. coli K1− strains. The fimH gene was present in all tested E. coli K1+ strains and in 97.0 % of non-K1 strains. E. coli K1+ strains were significantly more likely to possess the following genes than E. coli K1− strains: sfa/foc (37.3 vs 16.4 %, P = 0.006), ibeA (35.8 vs 4.5 %, P<0.001), iutA (82.1 vs 35.8 %, P<0.001) and hlyF (28.4 vs 6.0 %, P<0.001). In conclusion, E. coli K1+ seems to be more virulent than E. coli K1− strains in developing severe infections, thereby increasing possible sepsis or neonatal bacterial meningitis.

The use of antimicrobial agents in the management of patients infected with Shiga toxin-producing Escherichia coli (STEC) O157 : H7 remains controversial. Here, we examined the antibacterial efficacy of a natural product, ellagitannin from Quercus infectoria (Qi 4), against the organism in a murine model. Streptomycin-pretreated mice were orogastrically inoculated with 2×109 c.f.u. streptomycin-resistant E. coli O157 : H7. The results demonstrated a stable high level of STEC present in the faeces of the infected animals. The bacterial levels in infected mice receiving Qi 4 at MIC and 2×MIC were not significantly different from those of the untreated group (t-test; P>0.05). In contrast, Qi 4 at 4×MIC significantly reduced the numbers of STEC within 2 days (t-test; 0.05>P>0.01). No viable bacteria were detected between day 5 and day 10. Similarly, at day 10, no organisms were detected from the intestines of the Qi 4-treated group, while they were recovered at levels of 108–11 and 105–10 c.f.u. g−1 in the colons and caeca of the infected mice, respectively. Histopathological findings from the infected kidneys revealed a marked increase in the number of mesangial cells and mesangial matrix. Ultrastructural examination of the kidneys from the infected mice also demonstrated proliferation of mesangial cells and an increase in the mesangial matrix. Cellular injury of endothelial cells with irregular borders and cytoplasmic bleb formation were noted. In contrast, the effects were not observed in the animals treated with Qi 4. The results clearly indicated that administration of Qi 4 could effectively eradicate the colonization of STEC O157 : H7 in the intestinal tract of mice and prevent renal injury. This compound may be an alternative candidate for a therapeutic agent against infections caused by this dangerous organism.

The Beijing Mycobacterium tuberculosis family is widely distributed and is the most common M. tuberculosis strain in East Asia. The highly transmissible and predominant Beijing M. tuberculosis strain in Korea, M. tuberculosis K1, was characterized using an aerobic challenge mouse model and a latent tuberculosis model with M. tuberculosis H37Rv as a reference. M. tuberculosis K1 multiplied over ten times more rapidly than M. tuberculosis H37Rv during the early stage of infection and induced high levels of histopathology in the lung. Low levels of T helper cell (Th) Th1 [interferon (IFN)-γ, interleukin (IL)-12p40] and Th2 cytokines (IL-4, IL-10) were induced in the lungs of M. tuberculosis K1-infected mice. In the latent model, mice infected with M. tuberculosis K1 exhibited more frequent relapse from the latent state than did mice infected with M. tuberculosis H37Rv. In conclusion, M. tuberculosis K1, a prevalent Beijing strain in Korea, is expected to spread due to its rapid growth during the early stages of infection, low-level induction of the immune response and high relapse rates from a latent state.

Bacteria of the genus Corynebacterium are important primary and opportunistic pathogens. Many are zoonotic agents. In this report, phenotypic (API Coryne analysis), genetic (rpoB and 16S rRNA gene sequencing), and physical methods (MS) were used to distinguish the closely related diphtheroid species Corynebacterium ulcerans and Corynebacterium pseudotuberculosis, and to definitively diagnose Corynebacterium renale from cephalic implants of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques used in cognitive neuroscience research. Throat and cephalic implant cultures yielded 85 isolates from 43 macaques. Identification by API Coryne yielded C. ulcerans (n = 74), Corynebacterium pseudotuberculosis (n = 2), C. renale or most closely related to C. renale (n = 3), and commensals and opportunists (n = 6). The two isolates identified as C. pseudotuberculosis by API Coryne required genetic and MS analysis for accurate characterization as C. ulcerans. Of three isolates identified as C. renale by 16S rRNA gene sequencing, only one could be confirmed as such by API Coryne, rpoB gene sequencing and MS. This study emphasizes the importance of adjunct methods in identification of coryneforms and is the first isolation of C. renale from cephalic implants in macaques.

Although previous studies investigating the MALDI Biotyper database (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based identification) have proven its high accuracy for bacterial identification, the studies differed in sample preparation, number of replicates, quantity of shots and target types used. In particular, the score cut-off values of special importance for reliable species identification varied. The aim of the present study was to identify species-specific differences in the mean score values for staphylococci. Cut-off values recommended by the manufacturer were adapted using the 20th percentile to rule out unknown score-modifying factors, even though the specificity was high and the lowest cut-off values would also yield an accurate result. Whilst correct species diagnosis was obtained in 97.32 % of samples (1382/1420), only 220 of all duplicates (15.49 %) revealed a score of ≥2.3, whilst 968 (68.17 %) had a score between 2.0 and 2.299, and 194 (13.66 %) had a score of <2.0. Ten of 21 species had a calculated 20th percentile of <2.0 and one species of <1.7. In conclusion, the use of species-specific cut-off values improves the relative sensitivity of species identification in staphylococci.

Bacterial spores are of continuing interest to the food and medical industries. In efforts to eliminate bacterial spore contamination, a number of sporicidal agents have been developed. Most of these compounds must be used carefully in very specific circumstances as they are toxic to humans. The sporicidal activity of Akwaton, a polyhexamethylene-guanidine hydrochloride (PHMGH)-based disinfectant, was tested against Bacillus subtilis spores. PHMGH is a colourless, odourless, non-corrosive and non-irritating antimicrobial biocide of the guanidine family. Spores suspended in distilled water and spores placed on solid surfaces (stainless steel and glass) were used to determine the log10 reduction after exposure to varying concentrations of Akwaton. The minimum sporostatic concentration, the minimum sporicidal concentration and the time required for sporicidal activity corresponded to 0.06% (w/v), 0.08 % (w/v) and 8.5 min, respectively. Disinfectant concentrations of 0.24 % (w/v) and 0.44 % (w/v) killed all spores suspended in distilled water within 3 min and 90 s, respectively. The sporicidal activity against suspended spores was linearly dependent with respect to the concentration of PHMGH and contact time (y 3 min = 40x−1.6 and y 90 s = 20x−0.8 thus y 3 min = 2y 90 s). Spores placed on surfaces were more resistant to the effect of the disinfectant and the positive linear correlation between the sporicidal activity and concentration was not observed. The concentration required to kill all spores placed on a surface (stainless steel or glass) corresponded to 0.52 % (w/v) for 90 s of contact and 0.36 % (w/v) for 3 min. This study demonstrated that PHMGH is an effective sporicidal disinfectant with great potential for use in hospitals, laboratories, food industries and households.

The aim of this study was to investigate the molecular characteristics of induced vancomycin resistance in Staphylococcus haemolyticus. Autolytic properties and phenotypic characteristics of passage-selected vancomycin-resistant S. haemolyticus strains were examined. In addition, expression of autolysis-related genes (atl, lrgAB, sarA and lytS) was investigated using the RNase protection assay (RPA). The RPA results indicated that only the expression of the atl gene was significantly upregulated (2.5- to 6-fold increase) in vancomycin-intermediate and vancomycin-resistant strains. The vancomycin-resistant strains exhibited lower expression of murein hydrolase proteins and reduced autolytic activity compared with the parent strain. In addition, a reduced growth rate, cell wall thickening and higher survival rate in the presence of lysostaphin were observed in vancomycin-intermediate and vancomycin-resistant induced strains compared with the parent strain. In conclusion, altered autolytic properties, in particular upregulation of the atl gene, may contribute to vancomycin resistance in S. haemolyticus.

We identified a Moraxella catarrhalis strain with high-level resistance to azithromycin (MIC>256 mg l−1), NSH1, isolated from nasopharyngeal swab samples from an inpatient with acute bronchitis in a Japanese hospital in 2011 and determined its mechanism of macrolide–lincosamide resistance. Antimicrobial susceptibility of M. catarrhalis strains was determined using the Etest and agar dilution methods. Mutations in the four 23S rRNA alleles, the ribosomal proteins L4 and L22, and methylase genes erm(B) and erm(F) were tested by PCR and/or sequencing. The efflux system was examined using appropriate inhibitors. Transformation experiments were performed using DNA amplicons of the 23S rRNA gene of M. catarrhalis strain NSH1. This strain showed high-level resistance to erythromycin, clarithromycin, azithromycin, clindamycin (MICs>256 mg l−1) and josamycin (MIC = 128 mg l−1), and contained the A2058T mutation (Escherichia coli numbering) in four of the 23S rRNA alleles. Mutation of the ribosomal proteins and overproduction of the efflux system were not observed, and methylase genes were not detected. When amplified DNA containing the single A2058T mutation was transformed into M. catarrhalis strains, transformants with three A2058T-mutated 23S rRNA alleles showed high-level resistance to macrolide–lincosamide, similar to strain NSH1. In contrast, transformants with two A2058T-mutated 23S rRNA alleles showed low-level MICs (azithromycin: 0.38–0.5 mg l−1). Thus, a single A2058T mutation occurring in at least three 23S rRNA alleles confers high-level resistance to 14-, 15- and 16-membered macrolides and lincosamides in M. catarrhalis possessing four 23S rRNA alleles. This study represents the first evidence, to our knowledge, of this effect in M. catarrhalis.

Epstein–Barr virus (EBV) BamHI-A rightward transcripts (BARTs; also designated complementary strand transcripts or CSTs) have been demonstrated to contain several splicing forms in EBV-infected cells. To date, however, little is known about the actual full-length splicing form and its functions. In the present study, we proved that six forms of BARTs were present in EBV-positive cell lines and various tissue specimens with different EBV infection patterns. Of the BART-encoded genes, mRNA of four major splicing forms, including BARF0, RPMS1, RPMS1A and A73, were expressed in all EBV-infected cells. On the other hand, mRNA of two minor splicing forms, RK-BARF0 and RB3, was rarely detected, or if at all, at very low expression levels. Both RPMS1A and RPMS1 mRNA was transcribed at higher levels in EBV-infected cells. In particular, RPMS1 mRNA was expressed abundantly in epithelial carcinoma cells, including gastric carcinoma and nasopharyngeal carcinoma, in association with a lytic infection signal, BZLF1 mRNA. The four major splicing forms were expressed much less in B-cell lines with an integrated EBV genome than in those with episomal EBV genomes. These data indicate that at least six splicing forms can be expressed by EBV-infected cells or tissues, although the expression patterns or levels differ for different infection states such as lytic and latent infections.

Reduced vancomycin susceptibility in Staphylococcus aureus in many cases appears to be associated with changes in biological characteristics, including reduced coagulase activity, cell wall thickening, slow growth, smaller colonies, decreased pigment formation and less or no haemolysis. Whether these changes affect identification by routine methods has not been reported. In this study, 24 vancomycin-susceptibility-reduced coagulase-negative staphylococci (CoNS) strains (including 22 Staphylococcus haemolyticus strains and two Staphylococcus epidermidis strains) were retested by PCR-based detection of Staphylococcus aureus-specific genes (nuc, coa and 16S rRNA). The results showed that six isolates identified by conventional biochemical tests as S. haemolyticus contained nuc, coa and 16S rRNA genes. These six strains were serial-passaged daily on nutrient agar without vancomycin supplementation, and vancomycin-susceptible revertants were obtained after 15 continuous passages. Revertant isolates were coagulase-positive and were identified as S. aureus by automated testing methods. This suggests that biochemical changes in S. aureus strains with reduced vancomycin susceptibility should be highlighted and that the detection of these strains requires more attention and improved techniques.

An in-house IS6110 real-time PCR (IH IS6110), MTB Q-PCR Alert (Q-PCR) and GenoType MTBDRplus (MTBDR; Hain Lifescience) were compared for the direct detection of Mycobacterium tuberculosis complex (MTBC) in 87 specimens following automated NucliSENS easyMAG DNA extraction. This included 82 first smear-positive specimens and three smear-negative specimens. Another in-house real-time PCR with a Mycobacterium genus-specific probe for the internal transcribed spacer (ITS) region (IH ITS) was used to allow a full comparison with culture results. The sensitivities of IH IS6110, Q-PCR, MTBDR and IH ITS for MTBC detection were 100, 92, 87 and 87 %, respectively, compared with culture. Both IS6110-based real-time PCRs (in-house and Q-PCR) were similar in performance, with 91.2 % concordant results for MTBC detection. Inhibition rates were low, with zero to three specimens producing uninterpretable results. However, the Q-PCR failed to detect MTBC in five samples that were smear negative or had few acid-fast bacilli (one to 10 bacilli in 10 microscopic fields) detected by IH IS6110. IH ITS was the least sensitive assay but may be useful when used in conjunction with IS6110 PCR results to determine the presence of non-tuberculous mycobacteria in smear-negative specimens. None of the real-time PCR assays tested provided drug-resistance data. It was concluded that an IH IS6110 assay could easily be incorporated into the workflow of a diagnostic laboratory for rapid and accurate identification of MTBC from clinical specimens. The inclusion of an internal control and amplification of an ITS target enhance the diagnostic utility of the test.

Sweet’s syndrome or acute febrile neutrophilic dermatosis has been associated with underlying infection, malignancy, inflammatory disease and certain medications. The infection agents associated with this include Streptococcus species, Yersinia species, Chlamydia species, Salmonella species and Helicobacter pylori. We report a case of Sweet’s syndrome in a 73-year-old woman following a 2 week course of severe gastroenteritis caused by Campylobacter species. Histological examination of skin lesions showed marked inflammatory infiltrate throughout the dermis, composed of neutrophils and histiocytes. The patient was successfully treated with topical and systemic steroids. To date, this is the first case of Sweet’s syndrome to be reported linked to Campylobacter species to our knowledge.

Invasive infections caused by Brevundimonas vesicularis are very rare in humans. We experienced an unusual case of liver abscess due to B. vesicularis in an immunocompetent young male. The patient was successfully treated by liver abscess drainage and with antimicrobial therapy of ceftriaxone followed by ampicillin/sulbactam. The organism found in the aspiration culture of the abscess material was initially reported, by using a VITEK 2 system, as Sphingomonas paucimobilis. However, later, B. vesicularis was confirmed as the true pathogen through 16S rRNA gene sequencing. To our knowledge, this is the first case of liver abscess caused by B. vesicularis.