- Volume 60, Issue 2, 2011

Since persistent infection with high-risk human papillomavirus (HPV) is a known cause of high-grade cervical intraepithelial neoplasia and cervical cancer, several HPV DNA detection methods have been developed during the last decade. The Hybrid Capture II (HCII) assay, which allows detection of 13 high-risk HPVs, has been well validated; however, it does not provide any genotype-specific information. The oncogenic activity of HPV is dependent on its genotype. The prophylactic effects of HPV vaccines are based on L1 virus-like particles and are limited mainly to infections corresponding to the HPV type used to develop the immunogen. Therefore, accurate detection and genotyping are important for treatment as well as screening. A newly developed HPV genotyping system using a liquid bead array was evaluated with 286 cervical samples and the results were compared to two commercially available methods, i.e. the HCII and HPV DNA chip assays, and sequencing. The sensitivity for detection of high-risk HPV was 85.3 % (HCII), 94.7 % (DNA chip) and 99.0 % (liquid bead array). The liquid bead array showed almost perfect agreement (κ=0.95) with genotype information confirmed by sequencing, while substantial agreement (κ=0.8) was observed between DNA chip and sequencing. Furthermore, the liquid bead array had superior detection of 26 HPVs (16 high-risk and 10 low-risk types) and has proven to be as accurate as sequencing in identifying individual HPV types, even in cases with multiple HPV infections.

All Staphylococcus aureus isolates (n=31) that caused bacteraemia in a Spanish geriatric hospital during 1996–2006 were analysed by a simple, rapid and inexpensive PCR technique based on variations in the hsdS1 and hsdS2 genes encoding the sequence recognition subunits of the Sau1 restriction–modification (RM) system. An equal number of isolates collected from surgical wounds over the same time period (control group) were similarly characterized. The RM test allocated 75 % of the isolates to the six major clonal complexes (CC1, CC5, CC8, CC22, CC30 and CC45) for which it was developed. However, recognition of minor CCs and precise identification of the circulating clones required more powerful and comprehensive techniques such as spa typing and multilocus sequence typing (MLST), which are more demanding and expensive. The RM test is not intended to replace spa or MLST typing, but may be of use when time, technical and/or financial resources are limited. Overall, nine and seven CCs were detected in bloodstream and wound isolates, respectively. In both groups, CC5 was the most frequent (35.5 % each), followed by CC45 or CC8 (22.6 and 32.3 % of bloodstream and wound isolates, respectively). The frequency of meticillin resistance was lower in bloodstream (16.1 %) than in wound (51.6 %) isolates (P=0.0025). Among the former, sequence type (ST) 5-staphylococcal cassette chromosome mec (SCCmec) II, ST5-SCCmec IV, ST45-SCCmec IV and ST125-SCCmec IV (now dominant in Spanish hospitals) clones were found. Among the wound isolates, nine meticillin-resistant clones were represented, with three of them (ST125-SCCmec III, ST125-SCCmec V and ST14-SCCmec V) being newly described.

Lactobacillus jensenii, Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri and Lactobacillus vaginalis were identified by 16S rRNA gene sequencing as the predominant culturable vaginal Lactobacillus species in a group of South African women, comprising 24, 22, 10, 10 and 9 %, respectively. A significant effect of vaginal discharge syndrome (VDS) and bacterial vaginosis (BV) on the distribution of predominant Lactobacillus species was observed. Whilst L. crispatus isolates were almost equally distributed between individuals with and without VDS and were not significantly reduced in women with BV versus normal microflora, L. jensenii isolates were significantly reduced in women with VDS (P=0.022) and reduced in women with BV versus normal microflora (P=0.053). Unlike L. crispatus, L. jensenii isolates were also significantly reduced in women with BV-associated VDS versus women without VDS and with normal microflora (P=0.051). In addition, lysogeny was commonly observed for L. crispatus, with 77 % of isolates yielding phage particles with contractile and non-contractile tails. Only 29 % of L. jensenii isolates yielded phage particles, and these were visible as tailless or podo-like particles.

The aim of this study was to establish the importance of detecting fluoroquinolone (FQ) resistance in multidrug resistant (MDR) Mycobacterium tuberculosis, and to show the usefulness of a hybridization-based line probe assay (LiPA) for detecting gyrA mutations. Thirty-three MDR M. tuberculosis isolates were collected from a total of sixty MDR isolates identified in Japan over 6 months during a national surveillance study in 2002. Seventeen MDR isolates were collected by the National Center for Global Health and Medicine in Japan over 6 years from 2003 to 2008. These 50 isolates were examined for FQ susceptibility, and analysed by LiPA and gyrA sequencing. Among them, 22 (44 %) showed FQ resistance. All FQ-resistant isolates had at least one mutation in gyrA. The results of the LiPA were fully consistent with the DNA sequencing results. Given that on the basis of our results almost half of the MDR M. tuberculosis isolates in Japan might have resistance to FQ, it is important to monitor FQ resistance in patients with MDR tuberculosis (TB), as well as with drug-susceptible TB, prior to commencing treatment. For the detection of FQ resistance, LiPA is useful and can rapidly and efficiently assess FQ resistance.

This study analysed the presence, location and transferability of integrons and antibiotic resistance genes in 103 Shigella sonnei outbreak isolates and in 32 sporadic isolates from Taiwan. Multiple antimicrobial resistance was common in both outbreak (95 %) and sporadic (97 %) isolates. Class 1 integrons were present in 34 outbreak isolates (33 %) and in six sporadic isolates (19 %). This study is the first, to our knowledge, to identify an atypical sul3-associated class 1 integron carrying the estX-psp-aadA2-cmlA-aadA1-qacH cassette array in Shigella. Class 2 integrons carrying the dfr1-sat2-aadA1 cassette array were predominant in outbreak isolates (90 %) but were not present in sporadic isolates. Other antimicrobial resistance genes not associated with integrons were found to encode resistance to ampicillin (bla TEM), chloramphenicol (cat1), sulfonamide (sul2) and tetracycline (tetA and tetB). The most common plasmid size was 130 kb (observed in 43 and 97 % of 1998 outbreak and sporadic isolates, respectively). In conclusion, the plasmid location of resistance genes and horizontal plasmid transfer promote the spread of multiple resistance genes in outbreak and sporadic isolates of S. sonnei.

This study was planned to evaluate the efficacy of silver nitrate and gentamicin in the treatment of burn wound infection and to compare it with phage therapy using an isolated and well-characterized Klebsiella-specific phage, Kpn5. A full-thickness burn wound was induced in mice and infected with Klebsiella pneumoniae B5055 via the topical route. Different concentrations of silver nitrate or gentamicin were applied topically daily after establishment of infection. Phage Kpn5 mixed in hydrogel was also applied topically at an m.o.i. of 200 on the burn wound site. The efficacy of these antimicrobial agents was assessed on the basis of percentage survival of infected mice following treatment. The results showed that a single dose of phage Kpn5 resulted in a significant reduction in mortality (P<0.001). Daily applications of silver nitrate and gentamicin at 0.5 % and 1000 mg l−1, respectively, provided significant protection (P<0.001) compared to lower concentrations of the two agents. However, the level of protection given by these two agents was lower than that given by the phage therapy. The results strongly suggest that phage Kpn5 has therapeutic utility in treating burn wound infection in mice as a single topical application of this phage was able to rescue mice from infection caused by K. pneumoniae B5055 in comparison to multiple applications of silver nitrate and gentamicin.

Acinetobacter baumannii is a Gram-negative pathogenic bacterium that often exhibits a multidrug-resistant phenotype causing infections at various sites of the body and increasingly leading to septicaemic shock. This study evaluated the role of acriflavine, a frameshift mutagen, on the movement of insertion sequence ISAba1 in clinical isolates of A. baumannii, with the focus on changes in expression levels of the bla ADC and bla OXA-51-like genes. Resistance profiles were assessed with consideration of ISAba1 acting as a promoter upstream of the bla ADC or bla OXA-51-like gene. ISAba1 movement was observed in the acriflavine mutants Ab153M and Ab1225M. Ab153M exhibited an increase in the MIC values of carbapenems and ceftazidime, with ISAba1 gained upstream of the bla ADC and bla OXA-51-like genes, correlating with an increase in gene expression. Reduced expression of the 17, 23 and 25 kDa outer-membrane proteins (OMPs) was also observed in Ab153M. There was a significant decrease in MIC values of carbapenems with the loss of ISAba1 upstream of the bla ADC and bla OXA-51-like genes in strain Ab1225M, and a significant decrease in bla OXA-51-like gene expression and, to a lesser extent, in bla ADC expression. Ab1225M and a serially subcultured Ab1225 strain (Ab1225s) exhibited overexpression of the 17, 23, 25 and 27 kDa OMPs. There was a decrease in MIC values of the carbapenems and piperacillin/tazobactam but not of ceftazidime in Ab1225s, which had ISAba1 upstream of the bla ADC and bla OXA-51-like genes. A significant decrease in bla OXA-51-like expression was observed in Ab1225s, whereas the expression of bla ADC was similar to that in the Ab1225 parental strain. The attenuation in this strain may be due to overexpression of OMPs and it is clear that, even if ISAba1 is present upstream of an antibiotic resistance gene, it may not necessarily contribute towards the overexpression of antibiotic resistance genes (bla OXA-51-like in Ab1225s). Movement of the IS element within the A. baumannii chromosome may be an important regulatory mechanism employed by the bacterium under particular stress conditions, and the ability to upregulate the expression of antibiotic resistance genes is likely to be an important factor in the pathogenicity of this bacterium.

In developing countries, diarrhoeal diseases are one of the major causes of death in children under 5 years of age. It is known that diarrhoeagenic Escherichia coli (DEC) is an important aetiological agent of infantile diarrhoea in Nicaragua. However, there are no recent studies on antimicrobial resistance among intestinal E. coli isolates in Nicaraguan children. The aim of the present study was to determine the antimicrobial resistance pattern in a collection of 727 intestinal E. coli isolates from the faeces of children in León, Nicaragua, between March 2005 and September 2006. All samples had been screened previously for the presence of DEC by multiplex PCR. Three hundred and ninety-five non-DEC isolates (270 from children with diarrhoea and 125 from children without diarrhoea) and 332 DEC isolates (241 from children with diarrhoea and 91 from children without diarrhoea) were analysed in this study. In general, antimicrobial resistance among the 727 intestinal E. coli isolates was high for ampicillin (60 %), trimethoprim–sulfamethoxazole (64 %) and chloramphenicol (11 %). Among individual E. coli categories, enteroaggregative E. coli isolates from children with and without diarrhoea exhibited significantly higher levels of resistance (P<0.05) to ampicillin and trimethoprim–sulfamethoxazole compared to the other E. coli categories. Resistance to ceftazidime and/or ceftriaxone and a pattern of multi-resistance was related to CTX-M-5- or CTX-M-15-producing E. coli isolates. The results suggest that E. coli isolates from Nicaraguan children have not reached the high levels of resistance to the most common antibiotics used for diarrhoea treatment as in other countries.

Piperine, a major plant alkaloid found in black pepper (Piper nigrum) and long pepper (Piper longum), has shown potential for inhibiting the efflux pump (EP) of Staphylococcus aureus. In this study, a modulation assay showed that piperine could decrease the MIC of ethidium bromide (EtBr) twofold at 32 μg ml−1 and fourfold at 64 μg ml−1 against Mycobacterium smegmatis mc2 155 ATCC 700084. A real-time, 96-well plate fluorometric method was employed to evaluate the EP inhibition ability of piperine in M. smegmatis. Reserpine, chlorpromazine, verapamil and carbonyl cyanide m-chlorophenylhydrazone were used as positive controls. Piperine significantly enhanced accumulation and decreased the efflux of EtBr in M. smegmatis, which suggests that it has the ability to inhibit mycobacterial EPs.

Corynebacterium jeikeium, a member of the non-diphtheria corynebacteria, has been rarely reported as being responsible for cardiovascular-device infection. Here, we report what is believed to be the first case of C. jeikeium pacemaker infection associated with the presence of proteinase-3 antineutrophil cytoplasmic antibodies. The diagnosis was established based on the positivity of a single positive blood culture and led to pacemaker extraction. This observation highlights the difficulty in the diagnosis of cardiac-device infection in the presence of a single positive blood culture with a fastidious microorganism that could be considered as a contaminant. It also underscores the need for device extraction to ensure healing.

Rhodococcus erythropolis rarely causes infection in humans. We report the second case of R. erythropolis septicaemia in a 7-year-old child. However, to our knowledge it is the first case in a patient with acute lymphocytic leukaemia who had been undergoing chemotherapy. The identification was performed using 16S rRNA gene sequencing. Even though R. erythropolis is rarely associated with human infections, it should be considered as a potential causative agent of bacteraemia, rather than overlooked as a contaminant.

We present a case of soft tissue infection caused by the basidiomycete Phellinus undulatus. To our knowledge, this is the first reported case of human infection caused by this fungus. Definitive identification was only possible through molecular analysis as the isolate failed to produce any distinct morphological features in vitro.