- Volume 60, Issue 1, 2011

Coxiella burnetii is the causative agent of Q fever, a disease with a spectrum of presentations from the mild to fatal, including chronic sequelae. Since its discovery in 1935, it has been shown to infect a wide range of hosts, including humans. A recent outbreak in Europe reminds us that this is still a significant pathogen of concern, very transmissible and with a very low infectious dose. For these reasons it has also featured regularly on various threat lists, as it may be considered by the unscrupulous for use as a bioweapon. As an intracellular pathogen, it has remained an enigmatic organism due to the inability to culture it on laboratory media. As a result, interactions with the host have been difficult to elucidate and we still have a very limited understanding of the molecular mechanisms of virulence. However, two recent developments will open up our understanding of C. burnetii: the first axenic growth medium capable of supporting cell-free growth, and the production of the first isogenic mutant. We are approaching an exciting time for expanding our knowledge of this organism in the next few years.

This study aimed to evaluate the distribution of superantigen gene profiles and the presence of exfoliative toxin genes in community-acquired meticillin-resistant Staphylococcus aureus (CA-MRSA) isolated from Chinese children, and simultaneously to assess virulence gene profiles and genetic background. Of the CA-MRSA isolates, 88.9 % (88/99) harboured toxin genes, with sek as the most frequent toxin gene (62.6 %), followed by seq (61.6 %), seb (60.6 %) and sea (35.4 %). The eta gene was detected only in one ST398-IVa-spa t034 strain. The sed and etd genes were not found in any of the isolates tested. A total of 38 virulence genotypes were observed, of which the genotype seb-sek-seq (27.3 %, 24/88) comprised the majority, followed by sea-seb-sek-seq (18.2 %, 16/88). The enterotoxin gene cluster including seg-sei-sem-sen-seo-seu predominated at a rate of 15.1 %. The relationship among toxin genotypes, toxin genes encoding profiles of mobile genetic elements and genetic background was analysed. Among 66 clonal complex (CC) 59 isolates, 87.9 % (58/66) were positive for toxin genes, and 75.8 % (50/66) harboured the toxin gene combination seb-sek-seq. Among seb-sek-seq-positive CC59 strains, 42.0 % (21/50) also carried the sea gene. CC59 corresponded exclusively to accessory gene regulator 1 (agr-1). The data presented here enhance our current knowledge on the virulence determinants of CA-MRSA.

In support of the surveillance of pneumococcal infections in the era of conjugate vaccines, a sensitive and specific multiplex immunoassay using xMAP beads has been developed for direct detection of pneumococcal serotype-specific polysaccharides in clinical samples, particularly urine. The assay was tested on panels of spiked urine specimens, clinical urine specimens and bacterial isolates. Each of the 14 serotypes in the multiplex assay can be detected to 0.1 ng purified polysaccharide ml−1, or less. Testing of a panel of urine specimens from patients with culture-confirmed pneumococcal or non-pneumococcal disease indicated that the multiplex assay is both sensitive and specific. The correct pneumococcal serotype was identified directly from urine in 46/58 (79.3 %) patients who had a contemporaneous blood culture isolate of a multiplex assay serotype. Furthermore, the specificity of the assay on this panel of samples was 99.3 % (145/146). This multiplex assay could be useful, in conjunction with the pneumococcal screening test Binax NOW, in urine for diagnosis of pneumococcal disease and the identification of the aetiological serotype, and potentially be of benefit in culture-negative patients.

Meticillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen in Pakistan and is emerging in the community. This is one of the first reports of the prevalent genotypes of MRSA in both hospital and community settings in Pakistan. Isolates collected in 2006–2007 were characterized by PFGE, staphylococcal cassette chromosome mec (SCCmec) typing and multilocus sequence typing (MLST). PFGE identified nine pulsotypes, the majority of isolates belonging to pulsotypes A (n=70) and B (n=38), which were predominant among hospital-onset MRSA (HO-MRSA) and community-onset MRSA (CO-MRSA) isolates, respectively. Among the HO-MRSA isolates, variants of SCCmec type III were prevalent, whilst SCCmec type IV or variants were predominant in the CO-MRSA isolates. MLST identified two principal sequence types, ST8 and ST239. An association was observed between ST8, PFGE pulsotype B and SCCmec type IV in the CO-MRSA (ST8-MRSA-IV). Similarly, ST239, PFGE pulsotype A and SCCmec type III were associated with HO-MRSA (ST239-MRSA-III). Therefore, the prevalent genotypes circulating in Pakistan at the time of study were ST8-MRSA-IV and ST239-MRSA-III in the community and hospital settings, respectively. A set of HO-MRSA isolates collected in 1997 were characterized by PFGE and SCCmec typing for comparison. The isolates belonged to two PFGE pulsotypes (A, n=28; B, n=11) and contained just two SCCmec types. These results suggest that an increase in genetic diversity occurred over the period 1997–2007 as a result of either microevolution or the importation of strains from surrounding areas.

A total of 1485 adult ticks were collected from mammalian hosts in south-eastern Sardinia, Italy, during the years 2007–2008. Ticks were identified and tested by PCR analysis for presence of Rickettsia species of the spotted fever group, Ehrlichia canis, Anaplasma phagocytophilum, Coxiella burnetii, Bartonella species and Leishmania species. Among all tick species examined (Rhipicephalus sanguineus, Rhipicephalus turanicus, Rhipicephalus bursa, Rhipicephalus pusillus, Hyalomma marginatum marginatum, Haemaphysalis sulcata and Dermacentor marginatus), only Hyalomma marginatum marginatum produced negative results. A total of 22 pools belonging to the three tick species Rhipicephalus sanguineus (0.9 %), Rhipicephalus turanicus (4.5 %) and Rhipicephalus pusillus (100 %) were positive for Rickettsia species, while a total of five pools belonging to Rhipicephalus sanguineus (0.09 %), Haemaphysalis sulcata (16.7 %) and D. marginatus (7.8 %) were positive for E. canis. Five pools of Rhipicephalus turanicus (1.8 %) were positive for A. phagocytophilum. Positivity for C. burnetii was found in seven pools belonging to three tick species: Rhipicephalus sanguineus (0.5 %), Rhipicephalus turanicus (0.3 %) and Haemaphysalis sulcata (4.4 %). Finally, four pools belonging to Rhipicephalus sanguineus (0.09 %), Rhipicephalus turanicus (0.7 %) and Rhipicephalus bursa (1.1 %) were positive for Bartonella species. Leishmania species DNA was not detected in any of the tick pools examined. Data presented here increase our knowledge on tick-borne diseases in Sardinia, and provide a useful contribution to understanding their epidemiology.

Antibody responses can be useful markers of tuberculosis (TB) infection, especially in the screening of extra-pulmonary TB. MPT64 is an important antigen in Mycobacterium tuberculosis (MTB) infection and is used in serological diagnosis. However, large variability in the diagnostic accuracy of MPT64 as a serological tool has limited its application. Phage-displayed random peptide libraries have emerged as a powerful technique to select peptides (epitopes) or mimotopes that may serve as surrogate diagnostic markers in serological tests. In the present study, this method was employed to identify mimotopes of the MPT64 protein of MTB by screening a linear heptapeptide library with rabbit antibodies raised against MPT64 protein. Two antigenic mimotopes (M2 and M6) resembling B-cell epitopes of MPT64 were identified that bound the affinity purified anti-MPT64 polyclonal antibodies and competed with MPT64 for antibody binding. From the results of sequence alignment and a structure modelling figure of MPT64, the sequence of the 2nd to 5th amino acids (DSML) of M2 was totally consistent with the sequence of the 224th to 227th amino acids of MPT64 and the peptide is located on the surface of the space structure of MPT64, suggesting that it might be a linear epitope of MPT64. The recognition of both phage-displayed and synthetic peptides of M2 by the anti-MPT64 polyclonal antibodies also supported this. Although no recurring sequence and no analogue to MPT64 of M6 were found for sequence alignment, the recognition of both phage-displayed and synthetic peptides of M6 by the anti-MPT64 polyclonal antibodies indicated that it might be a mimotope of a conformational epitope of MPT64. According to the results of the reactivity of human sera with synthetic M2 and M6 peptides and MPT64, M2 showed a significantly higher AUC and sensitivity than M6 and MPT64, especially for the sera from sputum-negative TB patients, suggesting that the M2 mimotope may be useful in serological diagnostic testing for TB.

Our objective was to analyse phenotypic and genetic data of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae, Enterobacter cloacae, Escherichia coli and Serratia marcescens that cause infections in our hospital. Over a 3 year period, 342 randomly selected clinical Enterobacteriaceae isolates were tested for ESBL production and evaluated for the presence of the β-lactamase genes bla SHV, bla TEM, bla CTX-M and bla TLA-1. The antibiotic susceptibilities of these isolates were also determined, and the clonality of the isolates was assessed by PFGE. Based on our analyses, 33/92 (35.9 %) K. pneumoniae, 31/87 (35.6 %) Enterobacter cloacae, 24/80 (30 %) E. coli and 17/83 (20.5 %) S. marcescens were identified as ESBL producers. The presence of TEM, SHV or CTX ESBL types was detected in 99/105 (94 %) of the isolates. TLA-1 was not detected in any of the 105 isolates. The dominant ESBL types were bla SHV-5 (n=33), bla SHV12 (n=31) and bla CTX-M-15 (n=30). The predominant ESBL identified in E. coli and Enterobacter cloacae isolates was CTX-M-15, whereas in K. pneumoniae and S. marcescens the predominant types were SHV-12 and SHV-5, respectively. PFGE genotyping revealed two main genetic patterns in the K. pneumoniae isolates, types SHV-12 and TEM-1+SHV-5. An outbreak caused by Enterobacter cloacae SHV-5+CTX-M-15 was detected. In contrast, most ESBL-producing isolates of E. coli and S. marcescens did not have similar PFGE banding patterns and thus were not genetically similar. Enterobacteriaceae are a concern in our hospital, especially K. pneumoniae and Enterobacter cloacae. Our results confirm that the CTX-M-15 ESBL type has spread rapidly in the hospital, and thus requires careful monitoring.

One hundred and three Streptococcus pyogenes isolates recovered mainly from streptococcal throat infections in Lebanon were characterized by emm and PFGE typing. Thirty-three emm types and subtypes were detected among the isolates. PFGE was more discriminatory as a typing method. The prevalent emm types were emm1 (12.6 %), emm22 (8.7 %), emm28 (7.7 %), emm88 (7.7 %) and emm4 (6.8 %) and all isolates were susceptible to vancomycin and penicillin G. Ten per cent of the isolates were resistant to erythromycin and 3 % were resistant to erythromycin and clindamycin, showing the macrolide–lincosamide–streptogramin B phenotype. The emm sequences and PFGE pattern database that were generated in this study will serve as a basis for information for long-term evolutionary and epidemiological studies of local S. pyogenes recovered not only in Lebanon, but also in neighbouring countries.

A single subcutaneous (s.c.) infection with 1×107 c.f.u. GAS472, a group A streptococcus (GAS) serotype M1 strain isolated from the blood of a patient suffering from streptococcal toxic shock syndrome, led to severe damage of striated muscle layers in the feet of mast cell (MC)-deficient WBB6F1-KitW/KitW-v (W/Wv ) mice 72 h after infection. In contrast, no damage was recognized in striated muscle layers in the feet of the control WBB6F1-Kit +/+ (+/+) mice 72 h after infection. In addition, adoptively transferred MCs reduced progressive tissue necrosis of the feet of W/Wv mice after infection. However, there was no significant difference in the mortality rates between the W/Wv and +/+ mice, or between the human CD46-expressing transgenic (Tg) mouse bone marrow-derived cultured MC-reconstituted W/Wv and non-Tg mouse bone marrow-derived cultured MC-reconstituted W/Wv mice after infection. Consequently, although MCs can help to reduce the severity of necrosis of the feet caused by s.c. infection with GAS472, such reduction of tissue necrosis scarcely improves the mortality rates of these mice. Moreover, human CD46 does not play a crucial role in the MC-mediated innate immune defence against GAS infection.

We report a case of septicaemia with prolonged, refractory hypotension related to Moraxella osloensis isolated in a non-immunocompromised paediatric patient.

Bacillary angiomatosis is a well-recognized infection with cutaneous and systemic manifestations caused by Bartonella henselae or Bartonella quintana and occurs in immunocompromised patients. We report a case of B. quintana bacillary angiomatosis following fludarabine-based chemotherapy for chronic lymphocytic leukaemia that was refractory to standard treatment and was complicated by lymphadenopathy and osteomyelitis.