- Volume 61, Issue 4, 2012

The opportunistic nosocomial pathogen Acinetobacter baumannii is responsible for a growing number of infections; however, few of its potential virulence factors have been identified, and how this organism causes infection remains largely unknown. Bacterial biofilms are often an important component in infection and persistence but there is no conclusive evidence to link biofilm formation with virulence and severity of infection in Acinetobacter. To investigate this link, several clinical isolates were assessed in biofilm culture models and were tested for virulence in the insect model Galleria mellonella. In both systems, the profiles showed significant differences between strains, but no correlation was observed between virulence and the ability to form biofilms. In contrast, A. baumannii cells from a biofilm produced higher mortality rates than an equivalent number of planktonic cells. Relative to planktonic cells, A. baumannii biofilm cultures also showed reduced sensitivity to antibiotics normally used in the treatment of A. baumannii, especially colistin. This model, therefore, provides a suitable system to investigate the link between biofilm growth and various factors influencing virulence during A. baumannii infection.

Amplification and sequence analysis of the 16S rRNA gene can be applied to detect and identify bacteria in clinical samples. We examined 75 clinical samples (17 culture-positive, 58 culture-negative) prospectively by two different PCR protocols, amplifying either a single fragment (1343 bp) or two fragments (762/598 bp) of the 16S rRNA gene. The 1343 bp PCR and 762/598 bp PCRs detected and identified the bacterial 16S rRNA gene in 23 (31 %) and 38 (51 %) of the 75 samples, respectively. The 1343 bp PCR identified 19 of 23 (83 %) PCR-positive samples to species level while the 762/598 bp PCR identified 14 of 38 (37 %) bacterial 16S rRNA gene fragments to species level and 24 to the genus level only. Amplification of shorter fragments of the bacterial 16S rRNA gene (762 and 598 bp) resulted in a more sensitive assay; however, analysis of a large fragment (1343 bp) improved species discrimination. Although not statistically significant, the 762/598 bp PCR detected the bacterial 16S rRNA gene in more samples than the 1343 bp PCR, making it more likely to be a more suitable method for the primary detection of the bacterial 16S rRNA gene in the clinical setting. The 1343 bp PCR may be used in combination with the 762/598 bp PCR when identification of the bacterial rRNA gene to species level is required.

Aspergillus terreus is an emerging pathogen that mostly affects immunocompromised patients, causing infections that are often difficult to manage therapeutically. Current diagnostic strategies are limited to the detection of fungal growth using radiological methods or biopsy, which often does not enable species-specific identification. There is thus a critical need for diagnostic techniques to enable early and specific identification of the causative agent. In this study, we describe monoclonal antibodies (mAbs) developed to a previously described recombinant form of the haemolysin terrelysin. Sixteen hybridomas of various IgG isotypes were generated to the recombinant protein, of which seven demonstrated reactivity to the native protein in hyphal extracts. Cross-reactivity analysis using hyphal extracts from 29 fungal species, including 12 Aspergillus species and five strains of A. terreus, showed that three mAbs (13G10, 15B5 and 10G4) were A. terreus-specific. Epitope analysis demonstrated mAbs 13G10 and 10G4 recognize the same epitope, PSNEFE, while mAb 15B5 recognizes the epitope LYEGQFHS. Time-course studies showed that terrelysin expression was highest during early hyphal growth and dramatically decreased after mycelial expansion. Immunolocalization studies demonstrated that terrelysin was not only localized within the cytoplasm of hyphae but appeared to be more abundant at the hyphal tip. These findings were confirmed in cultures grown at room temperature as well as at 37 °C. Additionally, terrelysin was detected in the supernatant of A. terreus cultures. These observations suggest that terrelysin may be a candidate biomarker for A. terreus infection.

In this study, 76 French and Tunisian urogenital specimens were subjected to molecular typing by using the two main Mycoplasma genitalium molecular typing methods, the mgpB single nucleotide polymorphism (SNP) typing method and the combination analysis of a variable-number tandem-repeat (VNTR) marker in MG309 and mgpB SNP. Furthermore, we tried to develop a multiple-locus VNTR analysis (MLVA) method. The genome of M. genitalium G37T was analysed for VNTRs and four VNTRs were used for an MLVA. The method, applied directly on clinical specimens, was based on a genescan analysis of VNTR loci labelled with fluorescent dyes by using multiplex PCR and capillary electrophoresis. This method had a 1.00 diversity index (DI) while the mgpB SNP typing and the combination of MG309 and mgpB SNPs had DIs of 0.853 and 0.989, respectively. However, among the sets of two concurrent specimens, taken at the same time from the urogenital tracts of 12 patients, only nine had matching MLVA profiles, while the two other methods gave identical profiles for all specimens amplified, except for one set. Moreover, eight new sequence types were described with the mgpB SNP typing method. The three molecular typing methods revealed a genetic heterogeneity, suggesting that M. genitalium was endemic in France and Tunisia and that the infections were not due to the clonal dissemination of one strain. Comparison of the typing results obtained with the three methods showed that the MLVA assay seemed too discriminatory to be used in future studies of sexual networks of M. genitalium infection. According to the discriminatory power and the feasibility of each mgpB-based method, we recommend that the mgpB analysis be used for general epidemiological studies and that the combination of MG309-STR and mgpB SNP methods should be used for sexual-network studies of M. genitalium infection.

Campylobacter is an important cause of foodborne gastroenteritis. We determined the occurrence of Campylobacter jejuni and Campylobacter coli, using culture-based methods and PCRs targeting virulence-associated genes (VAGs) among children aged ≤14 years who were treated for diarrhoea at emergency rooms in northeastern Brazil. Genomic DNA was extracted directly from stool samples collected from 366 children. A questionnaire was also applied to qualify the clinical conditions presented by each child at the time of admission. C. jejuni and C. coli were detected in 16.4 % (60/366) and 1.4 % (5/366) of the diarrhoeal samples, respectively, by PCR, a much higher proportion than that detected by conventional methods. C. jejuni VAGs were detected in the following proportions of hipO-positive samples: ciaB, 95 % (57/60); dnaJ, 86.7 % (52/60); racR, 98.3 % (59/60); flaA, 80 % (48/60); pldA, 45 % (27/60); cdtABC, 95 % (57/60); and pVir 0 % (0/60). Particular symptoms, such as blood in faeces, vomiting, fever, and/or abdominal pain, were not associated with detection of C. jejuni nor were they associated with any particular VAG or combination of VAGs (P>0.05). C. jejuni and its VAGs were detected in a substantial proportion of the children admitted. Further efforts shall be directed towards elucidating whether these genetic factors or their expressed proteins play a role in Campylobacter pathogenesis.

Peritoneal tuberculosis (TB) is a considerable problem in certain developing nations. Current diagnostic tests for peritoneal TB are difficult and time-consuming. This study aimed to determine the effectiveness of an adenosine deaminase (ADA) assay and the QuantiFERON-Gold (QFT-G) assay in the rapid diagnosis of TB peritonitis. Forty-one patients with a presumptive diagnosis of TB peritonitis with ascites were admitted to Mansoura University Hospital and included in the study. Ascitic fluid and blood samples were collected from each patient. Fluid samples were examined biochemically (protein concentration), cytologically (white blood cell count) and microbiologically (Ziehl–Neelsen stain and TB culture in Löwenstein–Jensen media), and ADA levels were determined using colorimetry. Interferon-γ levels in whole-blood samples were measured using the QFT-G assay. Fourteen (34 %) patients received a final clinical diagnosis of TB peritonitis; these patients were subclassified as definite (positive culture for Mycobacterium tuberculosis; eight patients), highly probable (four patients) and probable (two patients) for TB peritonitis. Of the 14 patients with a final clinical diagnosis of TB peritonitis, 3 (21 %) tested positive using an acid-fast bacilli smear, which showed a sensitivity of 21 % and a specificity of 100 %. A receiver operating characteristic curve showed that a cut-off value of 35 IU l−1 for the ADA level produced the best results as a diagnostic test for TB peritonitis, yielding the following parameter values: sensitivity 100 %, specificity 92.6 %, positive predictive value (PPV) 87.5 % and negative predictive value (NPV) 100 %. The QFT-G assay yielded the following values: sensitivity 92.9 %, specificity 100 %, PPV 100 % and NPV 96.4 %. The ADA and QFT-G assays might be used to rapidly diagnose TB peritonitis and initiate prompt treatment while waiting for a final diagnosis using the standard culture approach.

Nucleoside analogues may represent good candidates for the discovery of new antibacterial agents, therefore, a library of adenosine analogues was assessed for their antibacterial activity, and the relationship between the structure and activity of these molecules was outlined. Antibacterial activity was evaluated against that of reference strains of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Escherichia coli and Pseudomonas aeruginosa. We tested 54 adenosine analogues, modified both at ribose and base moieties, including adenine and 1/3-deazaadenine derivatives substituted in the 2- and/or N 6-positions and bearing N-9 sugar moieties, such as ribose, 2′-deoxyribose, 3′-deoxyribose, 2′,3′-dideoxyribose or cycloalkyl groups like cyclopentane. The data obtained, MIC and minimal bactericidal concentrations demonstrated that the presence of bulky substituents such as cycloheptyl and cyclooctyl rings on the N 6-amino, together with a chlorine atom in the 2-position, conferred antibacterial activity against the Gram-positive group with MIC values ranging from 16 to 128 mg l−1. The intact sugar moiety seemed to be not essential for antimicrobial activity and nucleosides bearing deoxyribose or cyclopentyl groups associated with bulky substituents in N 6-position showed good antimicrobial properties. Furthermore, N-1 proved to be non-crucial and the 2-chloro-N 6-cyclooctyl-1-deaza-3′-deoxyadenosine and 2′,3′-dideoxyadenosine compounds were among the more active in the series with an MIC of 32 mg l−1 against Staph. aureus and Strep. pneumoniae. None of the analogues was active against the two Gram-negative species tested. Hence, adenosine derivatives bearing bulky substituents in the N 6-position may represent good lead compounds for the future discovery of a novel series of antibacterial agents.

The phenotypic resistance to ethambutol (EMB) in Mycobacterium tuberculosis with embB gene mutations is still unclear. This study was designed to better understand EMB resistance due to embB gene mutation. Sequencing analysis of the embB gene was performed for 124 EMB-susceptible and 93 EMB-resistant M. tuberculosis strains isolated from South Korea. The MIC was determined for EMB-susceptible M. tuberculosis strains with the embB mutation and wild-type on Löwenstein–Jenson (LJ) solid medium in duplicate. Two (2.8 %) of 72 pan-susceptible, two (9.1 %) of 22 any-drug-resistant but EMB-susceptible, nine (30.0 %) of 30 multidrug-resistant (MDR) but EMB-susceptible and 84 (90.3 %) of 93 EMB-resistant M. tuberculosis strains possessed embB mutations at various codons including 306, 319, 354, 360, 399, 405, 406, 459 and 497. Strains with embB mutations at codons 306, 354, 399, 405 and 497 had highly pronounced EMB resistance, while strains with mutations at codons 319 and 406 mutations were moderately resistant and those with an embB459 mutation were EMB-susceptible at the critical concentration (2.0 µg ml−1) on LJ solid medium. However, the mean MIC for strains with embB mutations (1.42 µg ml−1) was higher than that for strains without the embB mutation (1.0 µg ml−1) in EMB-susceptible M. tuberculosis isolates (P = 0.0052). Three novel embB mutations at codons 399, 405 and 459 were identified in this study. These results support the hypothesis that embB mutation except for a few specific mutation types may be the main cause of EMB resistance.

Listeria monocytogenes is an important foodborne human pathogen. Human infection is associated with high mortality rates. Epidemiological investigation and molecular subtyping can be useful in linking human illness with specific sources of infection. This retrospective study describes the use of PFGE to examine relationships of 222 isolates from human and non-human sources in Ireland. Human clinical isolates from other countries were also examined. Eight small clusters of human and non-human isolates (mostly serotype 4b) that were indistinguishable from one another were detected, suggesting potential sources for human infection. For non-human isolates, some PFGE types appeared to be exclusively associated with a single source, whereas other PFGE-types appeared to be more widely disseminated. Indistinguishable, or highly related clusters of isolates of Irish and non-Irish origin suggest that some PFGE patterns may be globally distributed.

Human bocavirus (HBoV) is a parvovirus with a possible aetiological role in respiratory disease that is currently under investigation. We detected HBoV1 in children and adults hospitalized with acute disease of the lower respiratory tract. HBoV genome was detected by PCR in nasopharyngeal swabs collected from 75 patients aged 0–89 years during 2010. HBoV was found in 17/75 (22.7 %) patients, 64.7 % of them infants younger than 1 year old and 29.4 % adults older than 30 years [the bimodal age distribution among HBoV-positive (HBoV+) patients was statistically significant, P<0.001]. Of all HBoV+ cases, 35.3 % were co-infected; all co-infections occurred in children (≤13 years old) and 83.3 % of them were HBoV-respiratory syncytial virus (RSV) co-infections. Among infants younger than 1 year, 50 % HBoV+ specimens were co-infected, all of them with RSV. The rate of co-infection in infants was significantly higher compared to the frequency of co-infection in the whole cohort (P = 0.003). The results suggest that HBoV1 is involved in acute respiratory disease. Interplay between HBoV1 and RSV cannot be discarded as a cause of elevated percentages of co-detections in infants.

We characterized two STEC O104 : H4 clinical isolates collected in Poland from a 7-year-old boy with haemolytic uraemic syndrome (HUS) and his nanny. This household outbreak began on 29 May 2011. Because of its time-frame, the outbreak was assumed to be part of the international STEC O104 : H4 outbreak that arose in Germany in May 2011. The two Polish isolates were Shiga-toxin-producing Escherichia coli (stx2 lpf) with enteroaggregative E. coli pathotype (aggR aap aggA), thereby sharing the unique virulence properties of the epidemic STEC O104 : H4 strain from the international outbreak. The Polish isolates were multi-drug resistant and carried bla TEM, strA, strB, tetA, sul1 and sul2 markers together with the bla CTX-M-15 gene for CTX-M-15 extended-spectrum β-lactamase. PFGE patterns and plasmid profiles of the Polish isolates and the epidemic STEC O104 : H4 strain corresponded closely. This finding suggested an epidemiological link between the Polish STEC O104 : H4 isolates and the international outbreak. Retrospective serological investigations proved person-to-person transmission of the epidemic STEC O104 : H4 strain from a father who had visited Dortmund, Germany, to his 7-year-old son in Giżycko, Poland. To the best of our knowledge, this is the first report of household transmission of Shiga-toxin-producing E. coli in Poland.

Helicobacter pylori infection is often acquired in childhood and can persist for life. Previous studies in adult patients have shown that H. pylori isolates from North American and European hosts express predominantly type 2 Lewis x (Lex) and Ley epitopes, while Asian strains have the capacity to express type 1 Lea and Leb structures. In order to understand the influence of environmental and host factors on the expression of Le antigens, we analysed 50 Greek H. pylori isolates from symptomatic children. Both CagA-positive and -negative strains were evaluated. The expression of Le antigens was determined by whole-cell indirect ELISA (WCE), and LPS profiles were assessed by gel electrophoresis and immunoblotting. Occurrence of Lex and/or Ley antigens was confirmed in 35 of the isolates (70 %) while 15 of the isolates were non-typable. It was found that 11 of the paediatric isolates had the propensity to express type 1 Leb blood-group antigen (22 %), a feature relatively uncommon in H. pylori isolates from adults. One strain expressed both Leb and Lea antigens. The majority of the isolates (49/50, 98 %) expressed α1,6-glucan, an antigenic non-Le determinant present in the outer core region of H. pylori LPS. All Lex- and Ley-expressing strains also carried a functional cag pathogenicity island-encoding a type IV secretion system, capable of translocating CagA protein, as well as the vacAs1 allele, suggesting that Lex and Ley epitopes may aid the persistence of more aggressive strains. No association between bacterial virulence characteristics and the histopathological observations was evident.

The rapid emergence and dissemination of carbapenemase-producing Enterobacter species and other members of the Enterobacteriaceae poses a considerable threat to the care of hospitalized patients and to public health. In this study, Enterobacter isolates demonstrating decreased susceptibility to carbapenems detected at the Division of Hygiene and Medical Microbiology, Innsbruck Medical University, between January 2006 and December 2010 were tested for bla VIM-1, bla NDM-1, bla IMP, bla KPC and bla OXA-48 using a multiplex PCR with published primers. PFGE was performed to determine the genetic relatedness. In total, 33 isolates (28 Enterobacter cloacae and 5 Enterobacter aerogenes) were collected during the study period. From 2006 to 2009, between two and seven isolates were found per year. In 2010, a significant increase of carbapenem-resistant strains was observed (n = 12). The bla VIM-1 gene was detected in all 28 isolates of E. cloacae. Typing of E. cloacae by PFGE revealed three distinct clusters, the biggest of which contained 18 isolates. These findings demonstrate the emergence of VIM-1-producing Enterobacter in Tyrol, western Austria. The clonal relationship confirms the risk of spread of these organisms and their possible persistence over time.