- Volume 60, Issue 11, 2011

The pathogenesis of the primary stage of lymphogranuloma venereum (LGV) is poorly understood. There is no skin cell model and LGV pathogenesis studies are therefore carried out on cells of different origin. Moreover, such studies usually use reference strains, which may have evolved over the years in culture. In this study, a model was developed in which Chlamydia trachomatis enters and grows in human keratinocytes at 37 and 33 °C. Keratinocytes were infected with fresh clinical isolates and the three LGV reference strains L1, L2 and L3. Growth was monitored for 5 days post-infection using fluorescence microscopy and image analysis software. Chlamydial replication was quicker at 37 than at 33 °C, despite 33 °C being the temperature of human skin. The serovar L2 reference strain grew significantly faster than the other strains, although the fresh clinical isolates were also serovar L2. When grown in keratinocytes at 33 °C, the L2 and L3 reference strains produced much larger inclusions than the other strains tested. This model, which utilizes keratinocytes, better simulates the conditions present at the initial site of infection in LGV than previously published literature, making it a useful tool for future LGV pathogenesis studies. In addition, the results indicate that fresh clinical isolates should be included in LGV pathogenesis studies.

Francisella tularensis is a highly virulent intracellular bacterium capable of rapid multiplication in phagocytic cells. Previous studies have revealed that activation of F. tularensis-infected macrophages leads to control of infection and reactive nitrogen and oxygen species make important contributions to the bacterial killing. We investigated the effects of adding S-nitroso-acetyl-penicillamine (SNAP), which generates nitric oxide, or 3-morpholinosydnonimine hydrochloride, which indirectly leads to formation of peroxynitrite, to J774 murine macrophage-like cell cultures infected with F. tularensis LVS. Addition of SNAP led to significantly increased colocalization between LAMP-1 and bacteria, indicating containment of F. tularensis in the phagosome within 2 h, although no killing occurred within 4 h. A specific inhibitory effect on bacterial transcription was observed since the gene encoding the global regulator MglA was inhibited 50–100-fold. F. tularensis-infected J774 cells were incapable of secreting TNF-α in response to Escherichia coli LPS but addition of SNAP almost completely reversed the suppression. Similarly, infection with an MglA mutant did not inhibit LPS-induced TNF-α secretion of J774 cells. Strong staining of nitrotyrosine was observed in SNAP-treated bacteria, and MS identified nitration of two ribosomal 50S proteins, a CBS domain pair protein and bacterioferritin. The results demonstrated that addition of SNAP initially did not affect the viability of intracellular F. tularensis LVS but led to containment of the bacteria in the phagosome. Moreover, the treatment resulted in modification by nitration of several F. tularensis proteins.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used in clinical microbiological laboratories to identify bacteria and fungi at a species level and to subtype them. The cfiA gene encoding the unique carbapenemases found in Bacteroides is restricted to division II Bacteroides fragilis strains. The aim of this study was to evaluate whether MALDI-TOF MS is suitable for differentiating B. fragilis strains which harbour the cfiA gene from those that do not. A well-defined collection of 40 B. fragilis isolates with known imipenem MICs (0.062–>32 mg l−1) were selected for this study. Twelve B. fragilis strains with known cfiA status, including NCTC 9343 (division I) and TAL3636 (division II), were measured by means of microflex LT MALDI-TOF MS and well-defined differences in mass spectra between the cfiA-positive and cfiA-negative strains were found in the interval 4000–5500 Da. A further 28 strains were selected for the blind measurements: 9 cfiA-positive clinical isolates with different imipenem MICs ranging between 0.06 and >32 mg l−1 (different expressions of the metallo-β-lactamase gene) were clearly separated from the 19 cfiA-negative isolates. The presence or absence of the selected peaks in all tested strains clearly differentiated the strains belonging to B. fragilis division I (cfiA-negative) or division II (cfiA-positive). These results suggest a realistic method for differentiating division II B. fragilis strains (harbouring the cfiA gene) and to determine them at a species level at the same time. Although not all cfiA-positive B. fragilis strains are resistant to carbapenems, they all have the possibility of becoming resistant to this group of antibiotics by acquisition of an appropriate IS element for full expression of the cfiA gene, leading to possible treatment failure.

In this study, six clinical isolates (two from blood, two from urine and one each from a bronchoalveolar lavage and a vaginal swab) were identified as Candida rugosa based on carbohydrate assimilation profiles using API 20C AUX and ID32 C kits (bioMérieux). Sequence analysis of the D1/D2 domain of the yeasts differentiated the isolates into two subgroups, A and B (three isolates per subgroup), which were closely related (99.1–99.6 % nucleotide similarity) to C. rugosa strain ATCC 10571. Compared with the C. rugosa type strain, the intergenic transcribed spacer (ITS) nucleotide similarity for subgroup A was only 89.2 % (29 mismatches and one deletion) and for subgroup B was 93.7 % (20 mismatches). All isolates grew green colonies on Oxoid Chromogenic Candida Agar, with darker pigmentation observed for subgroup A. All isolates were able to grow at 25–42 °C but not at 45 °C. The isolates had identical enzymic profiles, as determined by API ZYM (bioMérieux) analysis, and produced proteinase. High amphotericin MICs (≥1 µg ml−1) were noted for two isolates from each subgroup. Dose-dependent susceptibility to fluconazole (MIC 32 µg ml−1) was noted in a blood isolate. The biofilms of the isolates demonstrated increased resistance to amphotericin and fluconazole. The greater ITS sequence variability of subgroup A isolates is in support of this yeast being recognized as a distinct species; however, further verification using more sophisticated molecular approaches is required. A sequence comparison study suggested the association of subgroup A with environmental sources and subgroup B with clinical sources. Accurate identification and antifungal susceptibility testing of C. rugosa are important in view of its decreased susceptibility to amphotericin and fluconazole. The ITS region has been shown to be a valuable region for differentiation of closely related subgroups of C. rugosa.

To compare the diagnostic sensitivity and specificity of seven Cryptosporidium diagnostic assays used in the UK, results from 259 stool samples from patients with acute gastrointestinal symptoms were compared against a nominated gold standard (real-time PCR and oocyst detection). Of the 152 ‘true positives’, 80 were Cryptosporidium hominis, 68 Cryptosporidium parvum, two Cryptosporidium felis, one Cryptosporidium ubiquitum and one Cryptosporidium meleagridis. The Cryptosporidium spp. diagnostic sensitivities of three Cryptosporidium and Giardia combination enzyme immunoassays (EIA) coupled with confirmation of positive reactions were 91.4–93.4 %, whilst the sensitivity of auramine phenol microscopy was 92.1 % and that of immunofluorescence microscopy (IFM) was 97.4 %, all with overlapping 95 % confidence intervals. However, IFM was significantly more sensitive (P = 0.01, paired test of proportions). The sensitivity of modified Ziehl–Neelsen microscopy was 75.4 %, significantly lower than those for the other tests investigated, including an immunochromatographic lateral flow assay (ICLF) (84.9 %) (P = 0.0016). Specificities were 100 % when the ICLF and EIA test algorithms included confirmation of positive reactions; however, four positive EIA reactions were not confirmed for either parasite. There was no significant difference in the detection of C. parvum and C. hominis by each assay, but the detection of other Cryptosporidium spp. requires further investigation, as the numbers of samples were small. EIAs may be considered for diagnostic testing, subject to local validation, and diagnostic algorithms must include confirmation of positive reactions.

The exotoxin gene content was established for 62 Staphylococcus aureus isolates causing bloodstream (n = 31) and wound (n = 31) infections in geriatric patients attending a long-term care Spanish hospital from 1996 to 2006. Content was determined based on PCR screening of genes encoding five haemolysins, three exfoliatins, three leukotoxins and 21 pyrogenic toxin superantigens (PTSAgs), in addition to markers of genomic (νSaβ) and pathogenicity (SaPIs) islands. Exotoxin genes were abundant in both bloodstream (11–23 genes) and wound (8–19 genes) isolates, and they were arranged in 55 combinations with only two represented in both groups. All isolates were positive for genes encoding haemolysins (hl; 3–5) and PTSAgs (tst, se and sel; 5–14), whereas exfoliatin (et) and leukotoxin (luk) genes appeared in 98.4 and 51.6 % of isolates, respectively. The hlg, lukPV, tst, sec and selu genes were found significantly more frequently in bloodstream than in wound isolates, whereas hlg-variant, sea, seb, see and selk-selq were more frequent in wound isolates (P<0.05). Distinctive exotoxin gene combinations could potentially be associated with specific mobile genetic elements, including genomic islands [lukED, egc1 (seg, seln, sei, selm, selo) and egc2 (seg, seln, selu, sei, selm, selo)]; pathogenicity islands (etd, seb, sec, sell, selq, selk and tst); bacteriophages (eta, lukPV, sea, selp, selk, selq and see); and plasmids (sed, selj, ser, ses and set). The abundance of exotoxin genes and variety of arrangements shown by S. aureus from geriatric patients could play a role in the adaptation of the pathogen.

Meticillin-resistant Staphylococcus aureus (MRSA) has been recognized as one of the major pathogens in hospital as well as community settings. In India, the mean isolation rate of MRSA is 20–40 % and many studies have suggested an escalating rate of infections caused by this organism. Despite pharmaceutical and technological advancement, infections caused by MRSA still remain difficult to diagnose. The present study was undertaken to compare five phenotypic methods for the detection of MRSA. This involved examining 200 isolates of S. aureus by oxacillin disc diffusion, cefoxitin disc diffusion, oxacillin screen agar test, the latex agglutination test and growth on CHROMagar. PCR for mecA gene detection was taken as the gold standard. It was found that 35 % of all S. aureus infections were caused by MRSA. The cefoxitin disc diffusion method, as recommended by the Clinical and Laboratory Standards Institute, was found to be a reliable method for MRSA detection but it should be supplemented with some other method like latex agglutination, CHROMagar or oxacillin screen agar testing so that no MRSA is missed. We recommend that along with cefoxitin disc diffusion, another method, preferably latex agglutination, should be routinely used in all hospitals to detect MRSA.

In contrast to most modern pharmaceuticals, probiotics are used in many parts of the world with little or no research data on the complex system of interactions that each strain may elicit in the human body. Research on probiotics has recently become more significant, as probiotics have begun to be prescribed by clinicians as an alternative for some gut infections, especially when antibiotics are contraindicated. This study attempted to elucidate the inhibitory interaction between the Japanese probiotic strain Clostridium butyricum MIYAIRI 588 (CBM588) and the hospital pathogen Clostridium difficile, which is responsible for a large proportion of antibiotic-associated diarrhoea and colitis. CBM588 has previously shown effectiveness against C. difficile in vivo, and here it was found that the toxicity of C. difficile in in vitro co-culture with CBM588 was greatly decreased or absent. This was dependent on the inoculation ratio and was not accounted for by the small degree of growth and mRNA inhibition observed. CBM588 and its cell-free supernatant also had no effect on toxin already secreted into the culture medium, and culture of the two strains separated by a semi-permeable membrane resulted in loss of the inhibition. Therefore, it was concluded that the detoxification probably occurred by the inhibition of toxin protein production and that this required close proximity or contact between the two species. The low-pH conditions caused by organic acid secretion were also observed to have inhibitory effects on C. difficile growth, metabolism and toxicity.

This study focused on identifying possible new options derived from natural sources for the treatment of bacterial infections. Several natural products were investigated for their potential in modulating Shigella–host-cell interactions. The proliferation of Shigella sonnei was effectively inhibited inside HEp-2 cells in the presence of 4-methoxycinnamic acid and propolin D. Propolin D also significantly reduced the apoptosis of infected macrophage-like U937 cells and moderately reduced the secretion of interleukin (IL)-1β and IL-18, which probably resulted from the inhibition of invasion plasmid antigen B secretion by this compound. Further characterization showed that propolin D did not prevent escape of Shigella from phagocytic vacuoles, as evidenced by actin-based motility and by the fact that addition of chloroquine did not further reduce the number of intracellular c.f.u. The role of propolin D in modulating autophagy could not be established under the experimental conditions used. As these compounds had no direct anti-Shigella activity in vitro, it was concluded that these compounds modulated Shigella–host-cell interactions by targeting yet-to-be defined mechanisms that provide benefits to host cells.

Candida is an important opportunistic human fungal pathogen. Infections caused by Candida albicans are related to the formation of a biofilm. The biofilm enhances the resistance of the C. albicans defence system, increases its resistance to antifungal drugs and induces increased drug tolerance, making clinical care more challenging. The in vitro activity of cis-2-dodecenoic acid (BDSF; a diffusible signal factor from Burkholderia cenocepacia) and trans-2-dodecenoic acid (trans-BDSF) against C. albicans growth, germ-tube germination and biofilm formation was estimated by absorbance measurements and microscopic assessments. C. albicans biofilms were prepared using a static microtitre plate model. Quantitative analysis of biofilm formation was performed using a 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide reduction assay to evaluate the effect of different concentrations of BDSF and trans-BDSF at different stages of biofilm formation. Reductions in biofilm structure and formation were visualized by inverted microscopy. Real-time RT-PCR was employed to estimate the mRNA expression levels of the hyphae-specific genes HWP1 and ALS3. It was found that 30 µM of either BDSF or trans-BDSF reduced germ-tube formation by approximately 70 % without inhibiting yeast growth. Yeast growth was strongly repressed by the exogenous addition of 300 µM BDSF and trans-BDSF at 0 and 1 h after cell attachment, with biofilm formation being reduced by approximately 90 and 60 %, respectively. BDSF and trans-BDSF were more effective against biofilm formation than farnesol and the diffusible signal factor cis-11-methyl-2-dodecenoic acid. None of the four drugs was able to destroy pre-formed biofilms. Real-time RT-PCR analysis showed that HWP1 was downregulated by approximately 90 % and ALS3 was downregulated by 70–80 % by 60 µM BDSF and trans-BDSF, implying that BDSF and trans-BDSF block C. albicans biofilm formation by interfering with the morphological switch. These results suggest that BDSF and trans-BDSF are potentially useful therapeutic agents worthy of further study.

We studied 137 uropathogenic Escherichia coli (UPEC) isolates from hospitalized adult patients (Queensland, Australia) for their resistance to 17 antimicrobial agents using the calibrated dichotomous sensitivity method and the presence of class I, II and III integron-associated integrase (intI) genes, including functional class II intI2, as well as the presence of sul1, sul2 and sul3 genes, using PCR. Randomly amplified polymorphic DNA PCR, a high-resolution biochemical-fingerprinting method (PhP) and phylogenetic grouping were also used to identify the clonality of the sulphonamide-resistant isolates. One hundred and twenty (87.6 %) isolates were resistant to one or more of the tested antimicrobial drugs, with the highest resistance (70.1 %) observed against sulphafurazole (96 isolates). Of these, 84 (87.5 %) contained one or more sul alleles, with sul1 being the most common allele [occurring in 69 (72 %) isolates]. Only 38 of 69 (55.1 %) strains carrying the sul1 gene were positive for class I integrase. Our results indicate a high prevalence of sulphafurazole-resistant UPEC strains belonging to different clones among patients with urinary tract infection in Queensland, Australia. We also conclude that these strains carry predominantly a sul1 gene that is not commonly associated with the presence of class I integrase, indicating that it may be carried on either a bacterial chromosome or other genetic elements.

This study examined the pattern of colonization of the neonatal gut by aerobic Gram-negative bacilli (GNB) and evaluated the association between gut colonization and sepsis in the developing world. This deserves attention because of the high incidence of sepsis and the differences in hygienic environments in developing countries compared with the developed world. The study was carried out on neonates in a tertiary-care government hospital. Serial gut samples were analysed (gastric aspirates and stool samples) for GNB. Blood samples of cases showing clinical signs of sepsis were also analysed for septic screening and culture positivity. Antibiograms, serotyping and PFGE were carried out to evaluate the relatedness of the gut and blood isolates. A diverse array of GNB was isolated from the gut of the neonates, Klebsiella pneumoniae being most common, followed by Escherichia coli. The rate of isolation of GNB was consistently higher in stool samples compared with gastric aspirate samples. Colonization was influenced by a stay in the neonatal intensive care unit and by the prolonged use of a feeding tube. GNB were the cause of sepsis in the majority of cases, with K. pneumoniae being the most frequently isolated GNB from the blood. Acinetobacter baumannii, Escherichia coli, Enterobacter cloacae and Burkholderia cepacia were the other GNB recovered from the blood of the neonates. Neonates with GNB in the gut had a higher incidence of clinical sepsis than those without. In 50 % of cases, the genotypes of the organisms found in the blood were indistinguishable from their gut counterpart. These results show that the neonatal gut is colonized with a diverse array of GNB, and an association between gut colonization and neonatal sepsis was observed.

Coagulase-negative staphylococci (CoNS) have become increasingly recognized as important agents of nosocomial infection. One of the characteristics of CoNS is their resistance to multiple antimicrobial agents commonly used for the treatment of staphylococcal infections. CoNS strains (n = 745) isolated from a university teaching hospital in China between 2004 and 2009 were tested for antibiotic resistance. The antibiotics were placed into three categories based on resistance levels of the CoNS strains to these antibiotics: high resistance (resistance rate >70 %), including penicillin G, oxacillin and erythromycin; medium resistance (resistance rate between 30 and 70 %), including tetracycline, clindamycin, ciprofloxacin, trimethoprim/sulfamethoxazole and chloramphenicol; and low resistance (resistance rate <30 %), including rifampicin, ceftizoxime and gentamicin. We also found that the prevalence of strains non-susceptible to teicoplanin increased from 4.5 to 6.7 % between 2008 and 2009. A one-step vancomycin agar selection experiment and subsequent population analysis revealed potentially vancomycin-resistant subpopulations that have been selected from the teicoplanin-non-susceptible strains. Vigilant surveillance of nosocomial isolates of CoNS is needed to determine their resistance to glycopeptides.

Streptococcus suis, particularly serotype 2, is a pathogen of both pigs and humans associated with a wide range of diseases, including meningitis, septicaemia and endocarditis. Among the genes in the capsular polysaccharide biosynthesis (cps) locus, cps2J exists only in the serotype 2 and 1/2 strains; therefore, cps2J-positive strains are suspected to have capsules of serotype 2 or 1/2. Coagglutination using antiserotype 1 and antiserotype 2 sera and/or transmission electron microscopy analysis of 288 cps2J-positive isolates from pigs showed that 32 (100 %) isolates from meningitis were encapsulated, whereas 86 (34 %) of 256 isolates from endocarditis were unencapsulated, indicating that capsule loss often occurred in the isolates from endocarditis. To investigate the genetic backgrounds, we randomly selected 43 unencapsulated isolates and analysed their cps loci by PCR scanning. Among them, 8 and 10 isolates apparently had deletions and insertions, respectively, in cps loci. In addition, a representative unencapsulated isolate and an unencapsulated strain showed adherence to porcine and human platelets, a major virulence determinant for infective endocarditis, to a significantly greater extent than the encapsulated strains. Although the capsule is considered to be an important virulence factor in S. suis, these results suggest that loss of capsular production is beneficial to S. suis in the course of infective endocarditis.

Streptococcus mutans is one of the oral pathogens associated with infective endocarditis (IE). With respect to bacterial binding ability to the extracellular matrix, the Cnm protein, a cell surface collagen-binding adhesin of S. mutans, is known as one of the possible virulence factors with regard to IE. In this study, we aimed to determine the distribution of the cnm gene, which encodes Cnm, in a large number of clinical isolates of S. mutans from Thai subjects. Then, the cnm-positive strains were classified using a multilocus sequence typing (MLST) scheme, which we constructed previously. In addition, the data were analysed together with our previous MLST data of cnm-positive strains from Japan and Finland in order to evaluate the clonal relationship among S. mutans strains harbouring the cnm gene. The cnm gene was detected in 12.4 % of all 750 Thai isolates, and serotype f showed the highest rate of detection (54.5 %). According to the MLST data, two clonal complex groups were revealed as the important clones related to cnm-positive S. mutans from various origins of isolation. Moreover, the collagen-binding properties of S. mutans strains with the cnm gene were significantly greater than those of strains without the gene, although four cnm-negative strains classified into two sequence types (STs), ST110 and ST136, showed extremely high collagen-binding rates suggesting the presence of additional genes involved with collagen binding in these STs. Taken together, these results provided information on both epidemiological as well as evolutional aspects of S. mutans possessing the cnm gene.

We report a case of concurrent diphtheria and infectious mononucleosis in an 11-year-old Brazilian child. Two days after specific treatment for diphtheria was started the patient was discharged following clinical recovery. This case highlights the difficulties in the clinical diagnosis of diphtheria in partially immunized individuals, and for the management and control of diphtheria in developing countries.

‘Haemophilus quentini’ has been proposed as the name for a distinct and homogeneous Haemophilus genospecies associated with urogenital tract and neonatal-related infections. Reports of ‘H. quentini’ isolation from adult men are rare and the disease potential in this population is unknown. We report six cases where ‘H. quentini’ was isolated from the genito-urinary tract in males. The isolation of ‘H. quentini’ during routine urine and urethral culture in adult men may aid in the determination of unresolved urethritis and possible urinary tract infections.

Sneathia sanguinegens is an infrequent bacterium in clinical specimens. We describe a case of right elbow septic arthritis due to a Sneathia species most closely related to S. sanguinegens in a young immunocompetent woman. S. sanguinegens has never been implicated in osteoarticular infections.

We describe the success of adjunctive bacteriophage therapy for refractory Pseudomonas aeruginosa urinary tract infection in the context of bilateral ureteric stents and bladder ulceration, after repeated failure of antibiotics alone. No bacteriophage-resistant bacteria arose, and the kinetics of bacteriophage and bacteria in urine suggest self-sustaining and self-limiting infection.

We report a case of meningitis due to Streptococcus australis, a species of oral streptococcus. Accurate identification was performed by various molecular techniques.