- Volume 60, Issue 12, 2011

Pharyngitis caused by group A streptococci (GAS) is one of the most common infections around the world. However, relatively little is known about which genes are expressed and which genes regulate expression during acute infection. Due to their ability to provide genome-wide views of gene expression at one time, microarrays are increasingly being incorporated in GAS research. In this study, a novel electrochemical detection-based microarray was used to identify gene expression patterns among humans with culture-confirmed GAS pharyngitis. Using 14 samples (11 GAS-positive and three GAS-negative) obtained from subjects seen at the Brooke Army Medical Center paediatric clinic, this study demonstrated two different clusters of gene expression patterns. One cluster expressed a larger number of genes related to phages, immune-system evasion and survival among competing oral flora, signifying a potentially more virulent pattern of gene expression. The other cluster showed a greater number of genes related to nutrient acquisition and protein expression. This in vivo genome-wide analysis of GAS gene expression in humans with pharyngitis evaluated global gene expression in terms of virulence factors.

Clostridium perfringens type A is the causative agent of a variety of histotoxic and enteric diseases. The ability of C. perfringens spores to germinate in vivo might be due to the presence of nutrient germinants in the host tissue and blood. In the current study, we investigated the ability of spores of C. perfringens wild-type and mutation strains to germinate in blood. Results indicate that spores of all three surveyed C. perfringens wild-type isolates germinated better in blood than in brain heart infusion (BHI) broth. However, as expected, spores lacking germinant receptor (GR) protein GerAA or GerKB germinated like wild-type spores in BHI broth and blood. Strikingly, while spores lacking GR proteins GerKA and GerKC showed significantly decreased germination in BHI broth, these spores germinated well in blood, suggesting that blood factor(s) can trigger spore germination through a GR-independent pathway. Using C. perfringens spores lacking cortex lytic enzymes (ΔcspB or ΔsleC ΔsleM), we were able to identify a host serum germination factor with peptidoglycan hydrolysing activity that (i) restored the colony-forming efficiencies of ΔcspB and ΔsleC ΔsleM spores up to ~5–20 % of that of total colony-forming spores; (ii) increased the number of c.f.u. of decoated ΔcspB and ΔsleC ΔsleM spores to ~99 % of that of colony-forming spores; (iii) and finally lost enzymic activity after heat inactivation, consistent with serum germination factor being an enzyme. Further characterization demonstrated that serum germination factor is very likely lysozyme, which can form a stable high molecular mass complex of ~120 kDa in serum. In conclusion, the current study indicates that a host serum germination factor with peptidoglycan hydrolysing activity is capable of triggering germination of C. perfringens spores by directly degrading the spore peptidoglycan cortex. Collectively, this study contributes to our understanding of the mechanism of in vivo germination of spores of C. perfringens.

In this study 86 isolates of Vibrio cholerae were analysed for their adhesive properties and the presence of pathogenicity island genes. With the exception of three isolates, all of the other clinical isolates (92.5 %) contained an intact TCP (toxin-co-regulated pilus) gene cluster. In contrast, 95 % of all environmental non-O1-non-O139 isolates were negative for the TCP gene cluster. The majority of clinical isolates (82.5 %) possessed the complete vibrio pathogenicity island (VPI) gene cluster and had a similar RFLP pattern, while only a single environmental strain possessed an almost complete VPI cluster (lacking 0.4 kb in the tcpA and toxT region). The result showed that the isolates with tcpA+/toxT+ had a strong attachment for HT-29 and Vero cells, whereas isolates with tcpA +/toxT − or tcpA −/toxT − genomic characteristics showed no autoagglutination and weak attachment for the cell lines. Two environmental strains (tcpA−/toxT− ) showed strong adhesive properties to the cell lines, indicating that non-fimbrial adhesive factors are involved in the environmental V. cholerae strains in the absence of TCP.

Brucellosis is a worldwide zoonotic disease that often requires serology for diagnosis. The serum agglutination test is the gold standard assay, but ELISAs are used by many laboratories. Many commercial ELISAs are available, but few studies have compared their performance. This study compared the ability of four commercially available ELISA kits (from Bio-Quant, Immuno-Biological Laboratories – America, Vircell and Euroimmun) to diagnose brucellosis in patients from Egypt and the USA. The sensitivities for all kits tested, except the Vircell kit, were >90 %, whilst the specificities were variable, with the Bio-Quant assay having a specificity of <40 %. Detection of IgG antibody was more sensitive than detection of IgM antibody for diagnosing brucellosis cases, but the specificity was comparable. Overall, there was good agreement between all of the kits except for the Bio-Quant kit. None of the diagnostic assays was 100 % reliable for diagnosing brucellosis; therefore, serology results need to be considered in tandem with patient history, clinical signs and other test results.

Early detection of aetiological agents is pivotal for adequate therapy for bacterial infections. Although culture is still considered the mainstay for laboratory diagnosis, it often lacks sensitivity, especially in patients already treated with antibiotics. The present study investigated the potential clinical utility of the commercial real-time-PCR-based system SeptiFast (SF), originally intended for diagnosis of sepsis from blood specimens, in the aetiological diagnosis of other bacterial infections, in patients undergoing antibiotic therapy. A total of 53 non-blood specimens were analysed for microbial pathogen detection by conventional culture and with SF real-time PCR: 19 (35.8 %) synovial fluids, 9 (17.0 %) cardiac valve tissues and 25 (47.2 %) purulent exudates from various body sites. Overall, the number of specimens positive for a pathogen by SF (26/53; 49.1 %) was significantly greater (P = 0.001) than that of specimens positive by culture (10/53; 18.9 %). In particular, SF was superior to culture for pathogen detection in cardiac valve tissues and synovial fluids. The analysis of concordance showed a fair agreement between the two methods (kappa value = 0.314; 95 % confidence interval = 0.531–0.097). Even with the limitation of the low number of specimens, this study confirmed the great potential of diagnosing bacterial infections by a molecular approach, and indicates that the real-time PCR SF system can be used for specimens other than blood, from patients undergoing antibiotic treatment.

The development of gonococcal point-of-care tests (POCTs) has been challenging due to the relatively monomorphic nature of the Neisseria genus. The BioStar Optical ImmunoAssay (OIA) POCT for diagnosing Neisseria gonorrhoeae infection detects a specific epitope on the L7/L12 ribosomal protein, which reduces cross-reactivity with other neisseriae, and produces a highly specific test. A laboratory-based evaluation of this POCT was performed to determine its analytical sensitivity and specificity. A panel of N. gonorrhoeae (n = 158) and non-gonococcal Neisseria (n = 62) isolates were examined. The OIA GC POCT positively reacted with 99.4 % of N. gonorrhoeae isolates and produced no reaction with 88.7 % of non-gonococcal Neisseria isolates. It cross-reacted with six strains of N. meningitidis and one non-speciated Neisseria sp., but failed to produce a positive result with one isolate of N. gonorrhoeae. The OIA GC POCT required a bacterial suspension of ~6.4×105 c.f.u. N. gonorrhoeae ml−1 and ~6.2×106 c.f.u. N. meningitidis ml−1 to produce a reactive result. The OIA POCT detected the majority of N. gonorrhoeae (99.4 %) isolates examined.

The aim of this study was to investigate the reliability of disc diffusion testing with penicillin, erythromycin and ciprofloxacin within the viridans group streptococci (VGS). In total, the antibiotic susceptibilities of 167 VGS isolates were compared by standard disc diffusion and broth microdilution methods, and these phenotypic data were compared to the carriage of the respective gene resistance determinants [ermB and mefA/E (macrolides); QRDR, gyrA, gyrB, parC and parE (quinolones)]. Overall, there were 35 discrepancies [resistant by MIC and susceptible by zone diameter (21.0 %)] between MIC and disc diameter when penicillin susceptibility was interpreted by Clinical and Laboratory Standards Institute criteria. Scattergrams showed a bimodal distribution between non-susceptible and susceptible strains when erythromycin susceptibility was tested by both methods. Thirty-four (20.4 %) isolates were categorized as resistant by MIC breakpoints, while disc diameter defined these as having intermediate resistance. With ciprofloxacin, three isolates (1.8 %) showed minor discrepancies between MIC breakpoints and disc diameter. Isolates non-susceptible to all three antimicrobial agents tested were reliably distinguished from susceptible isolates by disc diffusion testing, except for the detection of low-level resistance to penicillin, where broth microdilution or an alternative quantitative MIC method should be used. Otherwise, we conclude that disc diffusion testing is a reliable method to detect strains of VGS non-susceptible to penicillin, erythromycin and ciprofloxacin, as demonstrated with their concordance to their gene resistance characteristics.

The anti-staphylococcal activity of an ethanol extract of Rhodomyrtus tomentosa and its pure compound, rhodomyrtone, as well as their effects on staphylococcal biofilm formation and biofilm-grown cells were assessed. MIC and minimal bactericidal concentration values of the ethanol extract and rhodomyrtone against planktonic cultures and biofilms of five clinical strains each of Staphylococcus aureus and Staphylococcus epidermidis, and American Type Culture Collection (ATCC) strains of both species, were 32–512 and 0.25–2 µg ml−1, respectively. Results from time–kill studies indicated that rhodomyrtone at a concentration of 4× MIC could reduce the number of Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis ATCC 35984 cells by 99.9 % within 3 and 13 h, respectively. The ability of rhodomyrtone and the ethanol extract to prevent biofilm formation and kill mature biofilms was assessed: both demonstrated better activity than vancomycin at inhibiting staphylococcal biofilm formation. In addition, the viability of 24 h and 5-day staphylococcal biofilm-grown cells decreased after treatment with the ethanol extract and rhodomyrtone. The ability to reduce biofilm formation and kill mature biofilms occurred in a dose-dependent manner. Scanning electron microscopy clearly confirmed that treatment with rhodomyrtone at 16× MIC could reduce 24 h biofilm formation and the numbers of staphylococci, whilst at 64× MIC this compound destroyed the organisms in the 5-day established biofilm. These results suggest that rhodomyrtone has the potential for further drug development for the treatment of biofilm-forming staphylococcal infections.

This study was conducted to detect and analyse the presence of plasmid-mediated quinolone resistance (PMQR) determinants [qnr, aac(6′)-Ib-cr and qepA] among Citrobacter freundii isolates from patients in Anhui province, PR China. During 2009–2010, 31 C. freundii strains were collected from various hospital units and patient specimens. Using PCR, qnr genes were detected in eight isolates, but aac(6′)-Ib-cr and qepA genes were not found. The genes qnrA1, qnrB1, qnrB2, qnrB4, qnrB10 and qnrB24 were present in 6.5, 3.2, 6.5, 3.2, 3.2 and 3.2 % of C. freundii isolates, respectively. A new subgene of qnrB variant (qnrB24) was found and identified for what we believe to be the first time. PFGE after XbaI digestion of genomic DNA indicated that qnr-positive strains were not clonally related. Conjugation experiments were conducted to determine whether the qnr-carrying plasmids were self-transferable, and plasmids of transconjugants were extracted and analysed. The qnr genes were transferred from three clinical isolates to their transconjugants. Two qnrA1 genes transferred quinolone resistance with a plasmid of ~11 kb, whilst the size of the plasmid carrying the qnrB4 gene was ~64 kb. The susceptibility of positive isolates and transconjugants was tested using an agar dilution method according to Clinical and Laboratory Standards Institute guidelines, and the MICs of ciprofloxacin and levofloxacin were determined using Etest strips. Most isolates with qnr genes were resistant to fluoroquinolones and other antimicrobial agents. The MICs of transconjugants showed reduced susceptibility to fluoroquinolones.

A relevant bacterial load in cutaneous wounds significantly interferes with the normal process of healing. Vitamin E (VE) is a known immunomodulator and immune enhancer. Here, it was shown that administration of VE before infection was effective at increasing the antimicrobial activity of daptomycin (DAP) or tigecycline (TIG) in a mouse model of wound infection caused by meticillin-resistant Staphylococcus aureus (MRSA). A wound was established through the panniculus carnosus of mice and inoculated with MRSA. Mice were assigned to six groups: a VE pre-treated group with no antibiotics given after MRSA challenge; two VE pre-treated groups with DAP or TIG given after MRSA challenge; two groups treated with DAP or TIG only after MRSA challenge; and a control group that did not receive any treatment. Mice receiving each antibiotic alone showed a 3 log decrease in the number of c.f.u. recovered compared with the control group, mice treated with VE plus TIG had a 4 log decrease, whilst mice treated with VE plus DAP had the largest decrease in c.f.u. recovered (5 logs). The increased antimicrobial effect seen from treatment with VE plus antibiotics was associated with increased levels of natural killer cell cytotoxicity, with a more pronounced increase in leukocyte populations in mice treated with VE plus DAP. These data suggest that treatment with VE prior to infection and subsequent antibiotic treatment act in synergy.

Carbapenem resistance in members of the Enterobacteriaceae is increasing. To evaluate the effects of tigecycline and polymyxin B against carbapenem-non-susceptible pathogens, 89 representative clinical carbapenem-non-susceptible Enterobacteriaceae isolates were recovered from seven hospitals from four cities in China during 2006–2009: 30 Serratia marcescens, 35 Klebsiella pneumoniae, seven Enterobacter cloacae, six Enterobacter aerogenes, five Escherichia coli, four Citrobacter freundii and two Klebsiella oxytoca isolates. Twenty-eight S. marcescens isolates were indistinguishable. The 35 K. pneumoniae isolates belonged to 12 clonal strains. Among the 89 Enterobacteriaceae isolates, 82 produced KPC-2, seven produced IMP (three produced KPC-2 simultaneously), three did not produce any carbapenemases and nine were deficient in porins. Polymyxin B was much more active than tigecycline against carbapenem-non-susceptible Enterobacteriaceae. The MIC50 and MIC90 of imipenem, meropenem, ertapenem, polymyxin B and tigecycline were 8 and 32 µg ml−1, 8 and 32 µg ml−1, 16 and 128 µg ml−1, 0.5 and 16 µg ml−1, and 4 and 16 µg ml−1, respectively. Rates of susceptibility to imipenem, meropenem, ertapenem and polymyxin B were 30.0 %, 27.5 %, 2.5 % and 89.2 % by CLSI criteria. The rate of susceptibility to tigecycline was 40 % and 17.5 % by Food and Drug Administration (MIC ≤2 µg ml−1) and European Committee on Antimicrobial Susceptibility Testing (MIC ≤1 µg ml−1) criteria, respectively. KPC-2- or IMP-producing E. coli transconjugants exhibited reduced susceptibility to carbapenems but were susceptible to polymyxin B and tigecycline with an MIC range of 0.5–2 µg ml−1, 0.25–2 µg ml−1, 0.5–4 µg ml−1, 0.5 µg ml−1 and 0.5–1 µg ml−1. In conclusion, carbapenem resistance in Enterobacteriaceae is mainly due to production of KPC-2, and polymyxin B is active for the carbapenem-resistant Enterobacteriaceae.

Arcanobacterium pyogenes is commonly isolated from ruminant animals as an opportunistic pathogen that co-infects with other bacteria, normally causing surface or internal abscesses. Twenty-eight strains of A. pyogenes isolated from forest musk deer suppurative samples were identified by their 16S rRNA gene sequences, and confirmed by amplification of the pyolysin-encoding gene (plo) in all isolates. The MICs of 14 commonly used antibiotics were determined by an agar dilution method. Class 1 and 2 intI genes were amplified to determine whether integrons were present in the A. pyogenes genome. Class 1 gene cassettes were detected by specific primers and analysed by sequencing. All of the strains were susceptible to most fluoroquinolone antibiotics; however, high resistance rates were observed for β-lactams and trimethoprim. A total of 18 of the isolates (64.3 %) were positive for the class 1 intI gene, and 16 (57.1 %) contained class 1 gene cassettes with the aacC, aadA1, aadA2, blaP1 and dfr2a genes. Most were present in the multi-resistant isolates, indicating a general concordance between the presence of gene cassettes and antibiotic resistance, and that the integrons have played an important role in the dissemination of antimicrobial resistance in this species.

The objective of this study was to screen for novel quorum-sensing inhibitors (QSIs) from traditional Chinese medicines (TCMs) that inhibit bacterial biofilm formation. Six of 46 active components found in TCMs were identified as putative QSIs based on molecular docking studies. Of these, three compounds inhibited biofilm formation by Pseudomonas aeruginosa and Stenotrophomonas maltophilia at a concentration of 200 µM. A fourth compound (emodin) significantly inhibited biofilm formation at 20 µM and induced proteolysis of the quorum-sensing signal receptor TraR in Escherichia coli at a concentration of 3–30 mM. Emodin also increased the activity of ampicillin against P. aeruginosa. Therefore, emodin might be suitable for development into an antivirulence and antibacterial agent.

Quinolone resistance in the family Enterobacteriaceae is mostly attributed to the accumulation of mutations in the bacterial enzymes targeted by fluoroquinolones: DNA gyrase and DNA topoisomerase IV. Here we isolated the Klebsiella pneumoniae strains KP3606 and KP4707 from different specimens from 2008 to 2010 in Taizhou Municipal Hospital of China, and discovered a new subtype qnrB31, for which the GenBank accession number is HQ418999, and another new subtype qnrB32, for which the GenBank accession number is HQ704413. Susceptibility testing showed that KP3606 had a reduced susceptibility (MIC ≥0.5 µg ml−1) to quinolones, while KP4707 was resistant to quinolones. Of all qnrB alleles, the novel variants the qnrB32 gene and qnrB31 gene have the highest amino acid identity. The results suggested that of all the various genes involved in resistance to quinolones, the qnrB gene is the most likely to be mutated, and plasmids might play a role in the dissemination and evolution of qnrB genes.