- Volume 62, Issue 3, 2013

Consumption of street food is considered a major health risk in the absence of public-health inspection programmes in Indonesia. It is hypothesized that ice used in street food could be one of the major sources of Vibrio cholerae contamination. This study documented V. cholerae contamination in edible ice from different areas of Jakarta, the capital city of Indonesia, and attempted to characterize the virulence potential of the strains. A selective medium was used to isolate 98 V. cholerae strains and their identity was confirmed using biochemical assays. Serological tests classified the majority (78 %) in the non-O1 serogroup. Multiplex PCR was used to detect the presence of V. cholerae virulence genes, namely ctxA, ompU, tcpA, ace, zot and toxR. The toxR, ctxA, ompU and zot genes were detected in 75, 26, 15 and 1 % of isolates, respectively. The ace and tcpA genes were not detected in any of the isolates. The ctxA gene encoding the cholera toxin subunit A, which has been associated only with clinical strains of O1, here was present in both serogroups. The antibiotic-resistance profile showed that 65, 60, 52, 39, 37, 19 and 3 % of the isolates were resistant to ampicillin, streptomycin, kanamycin, sulfamethoxazole–trimethoprim, erythromycin, tetracycline and ciprofloxacin, respectively. A large proportion of V. cholerae isolates came from west and south Jakarta, and these strains exhibited multidrug resistance to ampicillin, streptomycin, tetracycline, erythromycin, kanamycin and sulfamethoxazole–trimethoprim. Many of these isolates from west and south Jakarta also harboured toxR, encoding a regulator, and ctxA. The presence of multidrug-resistant V. cholerae with virulence genes in edible ice, which could cause a severe outbreak, reflects the poor water quality in Jakarta, and indicates an urgent need for better surveillance and management.

Streptococcus suis is an important agent of swine and human meningitis. Among several sequence types (STs) characterized within the S. suis strain population, ST7 has emerged only in China and has been reported to be the cause of the human outbreak caused by S. suis in 2005. S. suis ST7 was shown to be derived from S. suis ST1 through a single-nucleotide change in the housekeeping gene thyA. The virulence potential of S. suis ST7 is reported to be higher than that of the worldwide-studied pathogenic S. suis ST1. The pathogenesis of ST1 infection has been partially elucidated, but information on the pathogenesis of ST7 infections remains scarce. To improve our understanding of the mechanisms involved in the development of meningitis caused by ST7, this study compared the microglial inflammatory response induced by ST1 and ST7 strains. The data showed that S. suis ST7 possessed a higher ability to induce pro-inflammatory cytokine production and to activate mitogen-activated protein kinase pathways and several transcription factors. The stimulation of microglial cells by S. suis increased the expression levels of the nucleotide oligomerization domain 2 (Nod2) gene. Finally, the results indicated that signal transducer and activator of transcription 3 (STAT-3) was involved in the development of meningitis induced by S. suis ST7 infection.

Arthroderma benhamiae is a zoophilic dermatophyte belonging to the Trichophyton mentagrophytes species complex. Here, a population of A. benhamiae wild strains from the same geographical area (Switzerland) was studied by comparing their morphology, assessing their molecular variability using internal transcribed spacer (ITS) and 28S rRNA gene sequencing, and evaluating their interfertility. Sequencing of the ITS region and of part of the 28S rRNA gene revealed the existence of two infraspecific groups with markedly different colony phenotypes: white (group I) and yellow (group II), respectively. For all strains, the results of mating type identification by PCR, using HMG (high-mobility group) and α-box genes in the mating type locus as targets, were in total accordance with the results of mating type identification by strain confrontation experiments. White-phenotype strains were of mating type + (mt+) or mating type − (mt−), whilst yellow-phenotype strains were all mt−. White and yellow strains were found to produce fertile cleistothecia after mating with A. benhamiae reference tester strains, which belonged to a third group intermediate between groups I and II. However, no interfertility was observed between yellow strains and white strains of mt+. A significant result was that white strains of mt− were able to mate and produce fertile cleistothecia with the white A. benhamiae strain CBS 112371 (mt+), the genome of which has recently been sequenced and annotated. This finding should offer new tools for investigating the biology and genetics of dermatophytes using wild-type strains.

Biofilm resistance mechanisms are multifactorial and vary from one organism to another. The purpose of this study was to investigate the efficacy of linezolid against indwelling device-related meticillin-resistant Staphylococcus epidermidis (MRSE) biofilm, and compare this with other antimicrobials. MICs, minimum biofilm inhibitory concentrations (MBICs) and minimum biofilm eradication concentrations (MBECs) were determined by the microtitre plate method. Fourteen and thirteen isolates from patients with indwelling device-related bacteraemia (IDB) and indwelling device colonization not associated with bacteraemia, respectively, were assessed. High MBIC was associated with a high intensity of biofilm formation (gentamicin r = 0.796; linezolid r = 0.477; rifampicin r = 0.634; tigecycline r = 0.410; and vancomycin r = 0.771), but this correlation was not observed with MBEC. Linezolid demonstrated better in vitro antimicrobial activity than other antimicrobials (MBIC – gentamicin P<0.001, rifampicin P = 0.019, vancomycin P = 0.008; MBEC – gentamicin P<0.001, rifampicin P = 0.002, vancomycin P<0.001). Biofilm growth inhibition was strongly associated with biofilm formation intensity; however, biofilm eradication was not cell number dependent. MRSE biofilm eradication would represent a huge advance for IDB, although high concentrations of gentamicin, linezolid, rifampicin, tigecycline and vancomycin were required for that. In general, linezolid reached better in vitro concentrations and was demonstrated to be highly active against MRSE biofilms by inhibiting their growth during biofilm formation.

Multidrug efflux pumps, such as CmeABC and CmeDEF, are involved in the resistance of Campylobacter to a broad spectrum of antimicrobials. The aim of this study was to analyse the effects of two putative efflux-pump inducers, bile salts and sodium deoxycholate, on the resistance of Campylobacter to biocides (triclosan, benzalkonium chloride, chlorhexidine diacetate, cetylpyridinium chloride and trisodium phosphate), SDS and erythromycin. The involvement of the CmeABC and CmeDEF efflux pumps in this resistance was studied on the basis of the effects of bile salts and sodium deoxycholate in Campylobacter cmeB, cmeF and cmeR mutants. The genetic variation in the cmeB gene was also examined, to see whether this polymorphism is related to the function of the efflux pump. In 15 Campylobacter jejuni and 23 Campylobacter coli strains, bile salts and sodium deoxycholate increased the MICs of benzalkonium chloride, chlorhexidine diacetate, cetylpyridinium chloride and SDS, and decreased the MICs of triclosan, trisodium phosphate and erythromycin. Bile salts and sodium deoxycholate further decreased or increased the MICs of biocides and erythromycin in the cmeF and cmeR mutants. For cmeB polymorphisms, 17 different cmeB-specific PCR-RFLP patterns were identified: six within C. jejuni only, nine within C. coli only and two in both species. In conclusion, bile salts and sodium deoxycholate can increase or decrease bacterial resistance to structurally unrelated antimicrobials. The MIC increases in the cmeF and cmeR mutants indicated that at least one non-CmeABC efflux system is involved in resistance to biocides. These results indicate that the cmeB gene polymorphism identified is not associated with biocide and erythromycin resistance in Campylobacter.

This study investigated 147 multidrug-resistant Enterobacteriaceae and Pseudomonas aeruginosa isolates from hospitalized patients in Malaysia. Class 1 integrons were the most dominant class identified (45.6 %). Three isolates were shown to contain class 2 integrons (2.0 %), whilst one isolate harboured both class 1 and 2 integrons. No class 3 integrons were detected in this study. In addition, the sul1 gene was amplified in 35 % of isolates and was significantly associated with the presence of integrase genes in an integron structure. RFLP and DNA sequencing analyses revealed the presence of 19 different cassette arrays among the detected integrons. The most common gene cassettes were those encoding resistance towards aminoglycosides (aad) and trimethoprim (dfr). As far as is known, this study is the first to identify integron-carrying cassette arrays such as aadA2-linF, aacC3-cmlA5 and aacA4-catB8-aadA1 in the Malaysian population. Patients’ age was demonstrated as a significant risk factor for the acquisition of integrons (P = 0.028). Epidemiological typing using PFGE also demonstrated a clonal relationship among isolates carrying identical gene cassettes in Klebsiella pneumoniae and P. aeruginosa but not in Escherichia coli isolates.

Staphyloxanthin is the eponymous feature of the human pathogen Staphylococcus aureus, and the pigment promotes resistance to reactive oxygen species and host neutrophil-based killing. To probe the possible use of rhodomyrtone isolated from Rhodomyrtus tomentosa (Aiton) Hassk. leaves to inhibit pigment production in S. aureus, experiments were carried out to compare pigment production and the susceptibility of rhodomyrtone-treated S. aureus and untreated cells to oxidants in vitro. In addition, we observed the innate immune clearance of S. aureus after incubation with rhodomyrtone using an ex vivo assay system – human whole-blood survival. The results indicated that rhodomyrtone-treated S. aureus exhibited reduced pigmentation, and that rhodomyrtone treatment led to a dose-dependent increase in the susceptibility of the pathogen to H2O2 and singlet oxygen killing. Consequently, the survival ability of the treated organisms decreased in freshly isolated human whole blood due to less carotenoid pigment to act as an antioxidant scavenger. Rhodomyrtone may be acting via effects on DnaK and/or σB, resulting in many additional effects on bacterial virulence.

It is well known that carbapenem-resistant mutations in penicillin-binding proteins (PBPs) are not observed in most Gram-negative bacteria under either clinical or experimental conditions. To understand the mechanisms involved in carbapenem resistance, this study constructed a mutS- and tolC-deficient Escherichia coli strain, which was expected to have elevated mutation frequencies and to lack drug efflux. Using this mutant, carbapenem-resistant strains with target mutations were successfully and efficiently isolated. The mutations T547I/A, M574I and G601D were identified in the PBP2 gene. Meropenem (MEPM)-resistant strains with the PBP2 T547I mutation showed fourfold increased resistance to 1-β-methyl-substituted carbapenems, such as doripenem, MEPM and biapenem, but not to non-substituted carbapenems such as imipenem and panipenem and other β-lactams. In addition, resistance resulting from the G601D mutation was limited to MEPM, whilst the M574I mutation conferred resistance to MEPM, imipenem and panipenem. This is the first report, to the best of our knowledge, that E. coli also has a carbapenem-resistance mechanism as a result of PBP2 mutations, and it provides insight into the resistance profiles of PBP2 mutations to carbapenems with and without the 1-β-methyl group.

We examined the molecular characteristics of 13 phenotypically confirmed carbapenemase-positive Enterobacteriaceae clinical isolates, including the relationships between plasmid-mediated quinolone-resistance genes (qnr), 6′-N-aminoglycoside acetyltransferase-encoding genes [aac(6′)] and AmpC-encoding genes (pAmpC). Twelve isolates were bla IMP-1 positive (92.3 %), while one was bla IMP-11 positive (7.7 %). We detected qnr, aac(6′) and pAmpC genes designated bla ACT-1-like in 76.9 %, 100 % and 53.8 %, respectively, of the 13 isolates. Plasmids were transferred successfully for three of the 13 metallo-β-lactamase (MBL)-producing isolates, and the sizes of plasmids extracted from these donors and transconjugants were deduced to be 65 kb or 70 kb. OmpC or OmpF protein expression was reduced in all Enterobacter cloacae, and one Klebsiella oxytoca lacked OmpK36. We demonstrate what appears to be the first evidence that, in Japan, Enterobacteriaceae producing MBLs carry various plasmid-mediated resistance genes, which may cause a further decrease in carbapenem susceptibility through reduction of the expression of outer-membrane proteins.

As a superantigen, staphylococcal enterotoxin C2 (SEC2) has commonly been used as an antitumour immunotherapy agent in China. However, the clinical application of SEC2 has been hampered by its pyrogenic toxicity and the presence of neutralizing antibody in patients. Thus, an improvement in its superantigen-based immunotherapy is highly needed. In this study, a truncated SEC2 mutant, SEC(14–128), was constructed without the N-terminal 13 and C-terminal 111 aa. This mutant retained T-cell proliferation and antitumour activities in in vitro experiments. However, it induced a significantly decreased release of the main inflammatory cytokines inerleukin-2 and gamma interferon. Moreover, SEC(14–128) exhibited reduced toxicity and affinity to anti-SEC2 IgG compared with native SEC2. Based on the considerable antitumour activity and low toxicity, it is proposed that the mutant SEC(14–128) could be a potential candidate for cancer treatment.

The aim of this study was to investigate the diversity and composition of the intestinal microbiota of elderly subjects using a combination of culture-dependent techniques and 16S rRNA gene amplicon sequencing. The study was performed as part of the ELDERMET project, in which 368 faecal samples were assessed for viable numbers of Bifidobacterium spp., Lactobacillus spp. and Enterobacteriaceae on selective agar. However, the Bifidobacterium selective medium used also supported the growth of Clostridium perfringens, which appeared as distinct colonies and were subsequently characterized phenotypically and genotypically. All the isolates were confirmed as toxin biotype A producers. In addition, three isolates tested also had the genetic determinants for the β2 toxin. Of the 368 faecal samples assessed, C. perfringens was detected in 28 samples (7.6 %). Moreover, C. perfringens was observed in samples from subjects in all the residence locations assessed but was most prevalent in subjects from long-stay residential care, with 71.4 % of the samples (63.2 % of the subjects) being from this residence location, and with a shedding level in excess of 106 c.f.u. (g faeces)−1. Microbiota profiling revealed some significant compositional changes across both the family and genus taxonomic levels between the C. perfringens-positive and -negative datasets. Levels of culturable Bifidobacterium spp. and Lactobacillus spp. were significantly (P<0.05) lower in the C. perfringens-positive samples. Sequence-based methods also confirmed a significant difference in the Bifidobacterium spp. level (P<0.05) between both datasets. Taken together, these data suggest that a high viable count [>106 c.f.u. (g faeces)−1] of C. perfringens in stool samples may be indicative of a less healthy microbiota in the intestine of elderly people in long-stay residential care.

Brevundimonas diminuta is rarely associated with invasive infections. We report the case of a previously healthy young man with pleural effusion, in which B. diminuta was recovered but incorrectly identified as Kingella kingae when it was freshly isolated. Consequently, the misidentification resulted in initial treatment failure. The correct identification was achieved through further incubation, sequencing of the 16S rRNA gene and MS.

Streptococcus suis is an important swine pathogen worldwide, which can be transmitted to human beings by direct contact; therefore, S. suis infections occur mainly in people who handle pigs or pork. We present a case of a patient with S. suis meningitis who worked as a butcher in a meat processing plant for 5 years. The 35-year-old man was admitted to the Department of Infectious Diseases in T. Browicz Memorial Central Infectious Disease and Observation Hospital in Bydgoszcz, Poland, with suspected bacterial meningitis. According to his medical history, the patient had been injured during the processing of pork. A microbiological examination of the cerebrospinal fluid and blood revealed S. suis as a single aetiological factor of this infection. The patient was empirically administered cefotaxime (2.0 g at 8-h intervals) and penicillin (9 million U at 8-h intervals). The patient made a complete recovery and his inflammatory markers normalized. Only the hearing deficit of his right ear did not disappear. An otolaryngologist recommended a 4-week steroid therapy. The patient was not examined because he did not report to the clinic. To our knowledge this is the first described case of human meningitis caused by S. suis in Poland.