- Volume 62, Issue 5, 2013

Enteroaggregative Escherichia coli (EAEC) is an important agent that causes endemic and epidemic diarrhoeal diseases worldwide. Several EAEC virulence-related genes (VRGs) have been described but their role in the clinical outcome of infection is not completely defined. This study investigated the prevalence of EAEC and potential associations of its VRGs with risk of or protection from diarrhoeal diseases in children from urban communities in north-eastern Brazil. The case–control study included 166 children, who had their stools evaluated for the EAEC diagnostic genes (aaiC and aatA) using PCR. Positive samples were further analysed by multiplex PCR and identified 18 VRGs. EAEC was found in the same proportion in both groups (41 %). The plasmid-borne gene encoding a hexosyltransferase homologue (capU) was the most frequently detected (89.6 %), followed by dispersin protein (aap, 58.2 %) and EAEC HilA homologue (eilA, 57.8 %). The AAF/III fimbrial subunit (agg3A) gene was observed at lower frequency (1.5 %). Plasmid-encoded toxin (pet) or AAF/II fimbrial subunit (aafA) was associated significantly with disease. AAF/IV fimbrial subunit (agg4A) or hypothetical plasmid-encoded haemolysin (orf61) was detected significantly more in controls than in children with diarrhoea. In addition, one set of genes in combination, aaiC and agg3/4C but lacking agg4A and orf61, was associated with diarrhoea cases; and another one, orf61 in the absence of pet and aafA, was correlated with control children. These data confirm a high prevalence, endemicity and heterogeneity of EAEC strains in the developing urban areas of north-eastern Brazil. Statistical correlation between cases and controls was seen with either isolated or combined sets of genes, suggesting that the pathophysiology of EAEC infection involves a complex and dynamic modulation of several VRGs.

Species in the genus Pigmentiphaga are Gram-negative, catalase- and oxidase-positive rods derived exclusively to date from environmental sources. Features of strains most like Pigmentiphaga daeguensis or Pigmentiphaga kullae from a case of suppurative otitis media in a 6-year-old female post-transplant recipient and in a human stool sample are described.

The presence of integrons and the antibiotic susceptibility profiles of STEC strains isolated in Belgium were analysed. The collection contained 306 strains, of which 225 were human isolates and 81 originated from different food or animal sources. Integrons were detected by PCR in 7.5 % of the tested isolates and all were class 1 integrons. The integron-positive strains all belonged to the human collection. By RFLP, five different types (A, B, C, D, E) were distinguished. The antibiotic-resistance gene cassettes were identified by sequencing representatives of the five different types. Two types of gene cassettes were found in different combinations, one encoding resistance to streptomycin/spectinomycin and the other encoding resistance to trimethoprim. One of the gene cassettes present was the rarely detected aadA23, which was now apparently for the first time reported in Western Europe. Susceptibility profiling of the strains for 11 antibiotics was done by standard disc diffusion assays. Among the 23 integron-positive strains, 17 different antibiotic susceptibility profiles were found. In the 283 integron-negative strains, 24 different antibiotic susceptibility profiles were observed. The majority of these strains were susceptible to all tested antibiotics (n = 218, 77.0 %). The integron-positive strains were significantly more resistant to eight of the eleven tested antibiotics compared to the integron-negative strains (P<0.05). PFGE profiles of integron-positive strains within selected serogroups did not cluster together.

Candida species are responsible for many opportunistic fungal infections. Fluconazole is a well-tolerated antifungal drug, commonly used in the treatment of candidiasis. However, with fluconazole resistance ever increasing, rapid detection and antifungal susceptibility testing of Candida is imperative for proper patient treatment. This paper reports a cost-effective, simple and rapid chromogenic agar dilution method for simultaneous Candida species identification and fluconazole susceptibility testing. The results obtained by X-Plate Technology were in absolute concordance with standard microbroth dilution assays. Analysis of 1383 clinical patient samples with suspected vulvovaginal candidiasis revealed that this technology was able to detect and speciate the Candida isolate and determine the fluconazole susceptibility. The prevalence and susceptibility profiles of the clinical isolates using this method were highly similar to published reports using the microbroth dilution method.

Members of the genus Actinomyces, Gram-positive, non-spore-forming anaerobic bacteria, are normal inhabitants of the mucosal surfaces of the oral, gastrointestinal and genital tracts. Identification of these bacteria using conventional methods is generally difficult because of their complex transport and growth requirements and their fastidious and slow-growing nature. However, in recent years, the advancement of molecular techniques has provided much improved identification and differentiation of closely related Actinomyces species. The aim of the present study was to evaluate the efficacy of the PCR technique in the diagnosis of genital Actinomyces in comparison with culturing and Papanicolaou (Pap) smear microscopy. Multiple sampling was conducted from 200 women using smear microscopy, culturing and PCR. Cyto-brushes were smeared on glass slides and stained using the routine Pap technique. Culturing was performed from a sterile swab, and Actinomyces were determined using the BBL Crystal ANR ID kit. PCR was performed from a second swab, and the Actinomyces type was determined using type-specific primers designed in our laboratory. Only one vaginal fluid sample (0.5%) revealed Actinomyces-like organisms on Pap smear examination. Actinomyces were detected in nine samples (4.5%) using the BBL Crystal ANR ID kit. Using PCR, eight samples (4%) were found positive for Actinomyces. No specimens that gave positive results by Pap smear microscopy and culturing could be confirmed by PCR. Pap smear microscopy and culturing were both found to have zero sensitivity for Actinomyces. PCR appears to be a sensitive and reliable diagnostic method for the detection of Actinomyces, which are difficult to cultivate from genital samples. PCR can be used for diagnostic confirmation in cases diagnosed by conventional methods, to prevent false-positive results.

Shared strains of Pseudomonas aeruginosa are now well recognized in people with cystic fibrosis (CF), and suitable P. aeruginosa laboratory typing tools are pivotal to understanding their clinical significance and guiding infection control policies in CF clinics. We therefore compared a single-nucleotide polymorphism (SNP)-based typing method using Sequenom iPLEX matrix-assisted laser desorption ionization with time-of-flight mass spectrometry (MALDI-TOF MS) with typing methods used routinely by our laboratory. We analysed 617 P. aeruginosa isolates that included 561 isolates from CF patients collected between 2001 and 2009 in two Brisbane CF clinics and typed previously by enterobacterial repetitive intergenic consensus (ERIC)-PCR, as well as 56 isolates from non-CF patients analysed previously by multilocus sequence typing (MLST). The isolates were tested using a P. aeruginosa Sequenom iPLEX MALDI-TOF (PA iPLEX) method comprising two multiplex reactions, a 13-plex and an 8-plex, to characterize 20 SNPs from the P. aeruginosa housekeeping genes acsA, aroE, guaA, mutL, nuoD, ppsA and trpE. These 20 SNPs were employed previously in a real-time format involving 20 separate assays in our laboratory. The SNP analysis revealed 121 different SNP profiles for the 561 CF isolates. Overall, there was at least 96 % agreement between the ERIC-PCR and SNP analyses for all predominant shared strains among patients attending our CF clinics: AUST-01, AUST-02 and AUST-06. For the less frequently encountered shared strain AUST-07, 6/25 (24 %) ERIC-PCR profiles were misidentified initially as AUST-02 or as unique, illustrating the difficulty of gel-based analyses. SNP results for the 56 non-CF isolates were consistent with previous MLST data. Thus, the PA iPLEX format provides an attractive high-throughput alternative to ERIC-PCR for large-scale investigations of shared P. aeruginosa strains.

One hundred and twenty-one isolates of Stenotrophomonas maltophilia complex were collected from seven Korean hospitals. Species and groups were identified using partial gyrB gene sequences and antimicrobial susceptibility testing was performed using a broth microdilution method. Based on partial gyrB gene sequences, 118 isolates were identified as belonging to S. maltophilia complex, including S. maltophilia, S. pavanii, Pseudomonas beteli, P. geniculata and P. hibisciola. The S. maltophilia isolates were further divided into three groups, I to III. S. maltophilia groups II and III were clustered into clade A with S. pavanii and P. beteli; S. maltophilia group I was clustered into clade B with P. geniculata and P. hibisciola. For all S. maltophilia complex isolates, the resistance rate to trimethoprim/sulfamethoxazole (TMP/SMX) was very high (30.5 %). Antimicrobial resistance rates varied among species or groups of S. maltophilia complex. Isolates of clade A showed significantly lower antimicrobial resistance rates than those of clade B; while 25 % of clade A isolates were multidrug resistant, 46 % of clade B isolates were multidrug resistant (P = 0.001). The finding of high antimicrobial resistance rates, particularly to TMP/SMX, among S. maltophilia complex isolates from Korea, and the existence of distinct groups among the isolates, with differences in antimicrobial resistance rates, suggests consideration of alternative agents to TMP/SMX to treat S. maltophilia infections and indicates the importance of accurate identification for appropriate selection of treatment options.

Subinhibitory concentrations (subMICs) of antibiotics may alter bacterial surface properties and change microbial physiology. This study aimed to investigate the effect of a subMIC (⅛ MIC) of penicillin (PEN) and erythromycin (ERY) on bacterial morphology, haemagglutinating activity, cell-surface hydrophobicity (CSH) and biofilm formation on glass and polystyrene surfaces, as well as the distribution of cell-surface acidic anionic residues of Corynebacterium diphtheriae strains (HC01 tox − strain; CDC-E8392 and 241 tox + strains). All micro-organisms tested were susceptible to PEN and ERY. Growth in the presence of PEN induced bacterial filamentation, whereas subMIC of ERY caused cell-size reduction of strains 241 and CDC-E8392. Adherence to human erythrocytes was reduced after growth in the presence of ERY, while CSH was increased by a subMIC of both antibiotics in bacterial adherence to n-hexadecane assays. Conversely, antibiotic inhibition of biofilm formation was not observed. All strains enhanced biofilm formation on glass after treatment with ERY, while only strain 241 increased glass adherence after cultivation in the presence of PEN. Biofilm production on polystyrene surfaces was improved by ⅛ MIC of ERY. After growth in the presence of both antimicrobial agents, strains 241 and CDC-E8392 exhibited anionic surface charges with focal distribution. In conclusion, subMICs of PEN and ERY modified bacterial surface properties and enhanced not only biofilm formation but also cell-surface hydrophobicity. Antibiotic-induced biofilm formation may contribute to the inconsistent success of antimicrobial therapy for C. diphtheriae infections.

Multidrug-resistant organisms (MDROs) pose significant infection-control challenges in settings with high prevalence and limited isolation facilities. This observational study in an 800-bed hospital determined the prevalence, bacterial density and genetic relatedness of MDROs isolated from ward surfaces, medical devices and the hands of healthcare professionals. The targeted MDROs were meticillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), Escherichia coli and Klebsiella pneumoniae resistant to extended-spectrum cephalosporins, and carbapenem-resistant (CR) Acinetobacter baumannii. During a 2-month period, microbiological sampling and molecular typing were performed on environment isolates, clinical isolates and isolates recovered from the hands of healthcare professionals. The target MDROs were recovered from 79 % of sampled surfaces, predominantly MRSA (74 % of all tested surfaces) and CR A. baumannii (29 %) but also VRE (2 %) and K. pneumoniae (1 %). MRSA was recovered from most tested surfaces throughout the ward, whilst CR A. baumannii was significantly more likely to be recovered from near-patient surfaces. Hand sampling demonstrated infrequent recovery of MRSA (5 %), CR A. baumannii (1 %) and VRE (1 %). Molecular typing of the study isolates identified seven MRSA and five Acinetobacter clonal clusters, respectively, and typing identified similar strains from the environment, patients and hands. Thus, in a healthcare setting with endemic circulation of MDROs, MRSA and CR A. baumannii were the predominant organisms recovered from ward surfaces, with MRSA in particular demonstrating widespread environmental dissemination. Molecular typing demonstrated the presence of related strains in patients, in the environment and on the hands of healthcare workers.

Bacteraemia and endocarditis are the most frequently reported clinical infections due to Abiotrophia defectiva species. This species has been rarely implicated in infections in neutropenic patients. We report a rare case of long-term venous catheter-related infection caused by A. defectiva that occurred in a febrile child who had neutropenia and Langerhans’ cell histiocytosis.

Urinary tract infections (UTIs) caused by Haemophilus parainfluenzae represent a very small percentage of this kind of pathology in children, and it has scarcely been described in the medical literature. According to previous studies of over 800 urine samples in children under 15 years old, a decrease of 50 % (from 0.13 % to 0.07 %) is estimated in its occurrence over the last two decades. This can be explained by the early detection of UTIs and their early empirical treatment, because this micro-organism shows high sensitivity to antibiotics. Also, the culture media in which this bacterium grows are not included in most current protocols. Here we report a case of a UTI caused by H. parainfluenzae in a 4-year-old boy.