- Volume 61, Issue 5, 2012

Propionibacterium acnes, a commensal of human skin, is also an opportunistic pathogen of common acne and certain infectious diseases. However, it is still not obvious which strain is pathogenic for a certain infectious disease, and investigations to characterize pathogenic strains using molecular typing methods such as MLST using several housekeeping genes have been undertaken. However, to date, such analysis has focused mainly on strains isolated from Europeans, and it is unclear whether the clonal distribution in other parts of the world is similar. Here, we analysed 50 strains of P. acnes from healthy humans and patients with atopic dermatitis (AD) in Japan and utilized MLST of seven housekeeping genes to study their clonal patterns. The MLST successfully typed the strains into five types, IA, IB1, IB2, II and III. Strains that belonged to types IA, IB and II were common on the human skin of both populations (Europe and Japan), but this study demonstrated what we believe to be the first association of type III strains with human skin, existing on the skin of both the AD and non-AD population. These results indicate the global existence of type III strains on human skin.

Staphylococcus aureus is a pathogen that can be the cause of nosocomial infections, community-acquired diseases and food-borne disease outbreaks. A number of variable number tandem repeat loci have been reported by various groups for use in epidemiological studies and outbreak investigations. The aim of this study was to systematically evaluate a total of 18 commonly used loci with the same S. aureus population so that the properties of each locus could be compared and used for typing, and to develop a computer program enabling calculation of the Simpson index (SI) of all possible loci combinations so that an optimal combination of loci could be identified for multilocus variable-number tandem-repeat analysis (MLVA) typing. A collection of 160 S. aureus isolates from patients with sporadic food-borne illnesses, and pet animals, such as canine, feline and equine pets, with skin infections were used to assess the MLVA loci. The newly developed Optimal Combination Finder (OCF) computer program was used for the analysis and it was found that the minimal number of loci combinations that produced the same SI (0.999528) as that of the 18 loci combined was eight; SIRU05, SIRU07, SIRU13, SIRU15, SIRU21, fnbB, sdrD and clfB. This suggested that the optimal combination of eight loci could be used for the routine investigation and surveillance of future S. aureus outbreaks instead of using all 18 loci. In addition, the OCF software could be a useful tool for the development of typing schemes for other organisms.

Staphylococcus aureus strain type USA300 is an important human pathogen. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry has been used successfully for rapid identification of S. aureus at the genus and species levels. Here, it was hypothesized that MALDI-TOF could be used to identify USA300 organisms at the strain level. A genetic algorithm model using ClinProTools software (Bruker Daltonics) was built using 47 isolates of USA300 S. aureus and 77 non-USA300 S. aureus isolates. Three mass/charge peaks (5932, 6423 and 6592) were found to be discriminators between the groups of isolates. The model was validated using 224 test isolates: 197 of 224 test isolates were correctly classified as ‘USA300 family’ or ‘non-USA300’. The sensitivity of the model was 0.87, with a specificity of 0.89, positive likelihood ratio of 8.19 and negative likelihood ratio of 0.15. The three-peak intensity MALDI-TOF model designed and tested in the current study can be used to rapidly identify USA300 family S. aureus isolates with reasonable sensitivity and specificity.

The potential of incorporating a real-time PCR for amplification and detection of 16S rRNA gene signatures directly from clinical samples was assessed as a tool for diagnostics. Universal PCR primers spanning short variable regions (~500 bp) were optimized for real-time PCR and tested in comparison with a longer fragment (~1400 bp) generated from block-based amplification. Real-time PCR had improved sensitivity of detection (8 % increase), decreased amplification time and simplified downstream processing. The real-time PCR primers also offered an improvement in detection of bacteria from samples that demonstrated inhibition with the block-based primers and in the resolution of mixed-sequence traces. In addition to testing primer sensitivity, the effect of amplifying and sequencing two different variable regions of the 16S rRNA gene on organism identification was compared. By amplifying and sequencing a shorter variable region, the number of species that were identified to the species level was reduced by 18 %.

When thermal injury damages the skin, the physical barrier protecting underlying tissues from invading micro-organisms is compromised and the host’s immune system becomes supressed, facilitating colonization and infection of burn wounds with micro-organisms. Within the wound, bacteria often develop biofilms, which protect the bacteria from the immune response and enhance their resistance to antibiotics. As the prophylactic use of conventional antibiotics drives selection of drug-resistant strains, the use of novel agents to prevent biofilm formation by wound pathogens is essential. In the present study, we utilized our recently developed in vitro wound biofilm model to examine the antibiofilm activity of garlic (Allium sativum). Wound pathogens were inoculated on sterile cellulose discs, exposed to formulated garlic ointment (GarO) or ointment base, and incubated to allow biofilm development. Biofilms were quantified and visualized microscopically. GarO prevented biofilm development by Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae, and caused a 2–5 log reduction of the bioburden within Enterococcus faecalis biofilms. Additionally, GarO disrupted partially developed biofilms produced by S. aureus, S. epidermidis and A. baumannii. The antistaphylococcal activity of GarO was stable for over 3 months at room temperature. Thus, GarO could be used as a prophylactic therapy to prevent wound biofilms caused by both Gram-negative and Gram-positive bacteria from forming, and may be a potential therapy for disrupting established staphylococcal biofilms.

Three closely related Klebsiella pneumoniae strains isolated from the same patient harboured bla CTX-M-15, bla OXA-1, bla SHV-11, qnrS1, aac(6′)-Ib-cr, oqxAB, aac(3)-II and aph(3′)-Ia genes. Two of the isolates were recovered after treatment with meropenem and showed resistance to carbapenems. Sequencing of ompK35 and ompK36 porin genes of the carbapenem-resistant strains revealed the presence of premature stop codons in both, and OmpK35 and OmpK36 porins were not detected by SDS-PAGE. One carbepenem-resistant strain showed a high amount of LamB protein and did not express OmpK26 porin whereas the other strain expressed OmpK26 but not LamB. The lack of major porins apparently causes changes in the expression of other, specific, porins.

A child’s death due to pneumococcal meningitis after contracting the disease in an after-school programme prompted an investigation to assess nasopharyngeal (NP) carriage among her contacts. The serotype of the meningitis case isolate was determined, together with the serotypes of the NP specimens of contacts, comprising the case patient’s brother, the case patient’s after-school programme contacts and the brother’s day-care centre (DCC) contacts. NP swabs from 155 children and 69 adults were obtained. Real-time PCR and conventional multiplex PCR (CM-PCR) assays were used to detect pneumococcal carriage and determine serotypes. Broth-enriched culture of NP specimens followed by pneumococcal isolation and Quellung-based serotyping were also performed. DNA extracts prepared from cerebrospinal fluid of the index case and from the NP strain isolated from the brother and from one attendee of the brother’s DCC were subjected to genotyping. Pneumococcal carriage assessed by real-time PCR and culture was 49.6 and 36.6 %, respectively (P<0.05). Twenty-three serotypes were detected using CM-PCR, with serotypes 6A/6B, 14, 19F, 6C/6D, 22F/22A, 23F and 11A/11D being the most frequent. All eight serotype 22F/22A NP specimens recovered were from children attending the brother’s DCC. The meningitis case isolate and the NP carriage isolate from the patient’s brother were both serotype 22F and shared the same new multilocus sequence type (ST6403) with the attendee of the brother’s DCC. CM-PCR proved to be useful for assessing carriage serotype distribution in a setting of high-risk pneumococcal transmission. The causal serotype appeared to be linked to the brother of the case patient and attendees of his DCC.

The objective of the current study was to determine whether the antimicrobial susceptibility profile or genotype of hospital-acquired isolates of Clostridium difficile differed from isolates causing community-acquired disease. Five hundred diarrhoeal stool samples (one >2 ml sample per patient) from patients across Manitoba, Canada, in 2006–2007 that were reported as C. difficile toxin positive were cultured, resulting in 432 isolates of toxin-positive C. difficile for analysis. Of these 432 isolates, acquisition status could be determined for 235 (54.4 %); 182 (77.4 %) isolates were hospital acquired and 53 (22.6 %) were community acquired. North American pulsotype (NAP) designations based on SmaI PFGE could be defined for 52.3 % of the 432 isolates, with NAP2 (n = 122) being the most common. Ninety-one per cent (71/78) of NAP2 isolates were recovered from patients with hospital-acquired C. difficile disease. Other NAP types and isolates with non-NAP-type PFGE patterns were less frequently associated with hospital-acquired disease. Community-acquired disease (35.3 % of isolates) was associated with a wide variety of NAP types. NAP2 isolates were homogeneous (85.5 % had SmaI PFGE pattern 0003) and demonstrated low susceptibility to moxifloxacin (6.6 %) and clindamycin (1.6 %) compared with non-NAP2 isolates (64.1–93.2 % moxifloxacin susceptible; 14.1–28.2 % clindamycin susceptible). All isolates of C. difficile in Manitoba were susceptible to metronidazole, piperacillin–tazobactam, amoxicillin–clavulanate and meropenem. NAP2 isolates of toxigenic C. difficile were approximately three times more common than NAP1 isolates (28.2 vs 9.1 %) in Manitoba in 2006–2007, and these isolates demonstrated high levels of clonality and multidrug resistance, and were associated with hospital acquisition.

Chlamydophila pneumoniae infection has been suggested to be associated with severe asthma characterized by persistent airway limitation, which may be related to airway remodelling. We investigated whether C. pneumoniae infection affected the secretion of metalloproteinase-9 (MMP9) and tissue inhibitor metalloproteinase-1 (TIMP1), and altered the responsiveness of inflammatory cells to corticosteroids. Human peripheral blood mononuclear cells (PBMCs) were cultured in vitro in the presence or absence of C. pneumoniae. Secretion of both MMP9 and TIMP1 was strongly suppressed by dexamethasone treatment in uninfected cells. MMP9 secretion was also significantly inhibited by dexamethasone in C. pneumoniae-infected cells, but TIMP1 secretion was not; hence the MMP9 to TIMP1 ratio decreased. Interestingly, expression of human glucocorticoid receptor β, which is believed to confer resistance to corticosteroids, was enhanced by dexamethasone treatment in C. pneumoniae-infected PBMCs. We conclude that C. pneumoniae infection may promote airway remodelling by decreasing the ratio of MMP9 to TIMP1 secreted by inflammatory cells, and by altering cellular responsiveness to corticosteroids.

The objectives of the study were to investigate the distribution of cphA-related genes (cphA) encoding a CphA metallo-β-lactamase (MBL) among 51 consecutive Aeromonas blood isolates and to compare different phenotypic methods for detecting CphA. The presence of cphA was detected by PCR. Four phenotypic methods, the imipenem-EDTA combined disc test, imipenem-EDTA MBL Etest, agar dilution test and modified Hodge test (MHT), were used to detect imipenem susceptibility and MBL production. The results showed that 35 (69 %) blood isolates had cphA. All (100 %) of 16 Aeromonas aquariorum isolates and 12 Aeromonas veronii isolates, and 4 (80 %) of 5 Aeromonas hydrophila isolates, carried cphA, but none of 15 Aeromonas caviae isolates did. With the standard inocula, irrespective of the presence or absence of cphA, all but one (50, 98 %) isolates were susceptible to imipenem tested by disc diffusion, Etest and agar dilution (104 c.f.u. spot inocula), and did not exhibit MBL production by the imipenem-EDTA combined disc test and MBL Etest. By the agar dilution test using large inocula (107 c.f.u.), 34 (97 %) of 35 cphA+ isolates had imipenem MICs of ≥16 µg ml−1, higher than the susceptible breakpoint (4 µg ml−1), and demonstrated positive results for the MHT, while one cphA+ and all 17 cphA− isolates had imipenem MICs of ≤4 µg ml−1. In conclusion, the distribution of cphA among aeromonads is species-specific, found in A. aquariorum, A. veronii and A. hydrophila, and the MHT may be a phenotypic screening test for CphA production.

Bacillus cereus is a well-known cause of foodborne disease usually of benign course. Here, we present the case of a 15-year-old boy who developed reversible fulminant liver failure associated with rhabdomyolysis after pasta consumption. Suspecting B. cereus as the aetiological agent may prevent unnecessary liver transplantation.

We describe a case of gastroenteritis caused by Campylobacter concisus. The pathogenic potential of C. concisus has yet to be elucidated. Recent studies indicate an association with enteric disease in immunocompromised patients and inflammatory bowel disease in children. Molecular identification methods may be necessary for identifying certain Campylobacter species because of phenotypic similarity.