- Volume 62, Issue 12, 2013

Listeria monocytogenes is an important food-borne bacterial pathogen and listeriosis can result in abortions in pregnant women. The bacterium can colonize food-processing environments, where specific molecular subtypes can persist for years. The purpose of this study was to determine the virulence potential of a group of food-processing persistent L. monocytogenes strains encoding a premature stop codon in inlA (encoding internalin A) by using two orally dosed models, pregnant mice and pregnant guinea pigs. A food-processing persistent strain of L. monocytogenes invaded placentas (n = 58; 10 % positive) and fetuses (3 % positive) of pregnant mice (n = 9 animals per strain), similar to a genetically manipulated murinized strain, EGD-e InlA m* (n = 61; 3 and 2 %, respectively). In pregnant guinea pigs (n = 9 animals per bacterial strain), a maternofetal strain (from a human fetal clinical fatal case) was isolated from 34 % of placenta samples (n = 50), whereas both food-processing persistent strains were found in 5 % of placenta samples (n = 36 or 37). One of the food-processing persistent strains, N53-1, was found in up to 8 % of guinea pig fetal liver and brain samples, whereas the maternofetal control was found in 6 % of fetal tissue samples. As the food-processing persistent strains carry a premature stop codon in inlA but are invasive in orally dosed pregnant mice and guinea pigs, we hypothesize that listerial crossing of the placental barrier can occur by a mechanism that is independent of an interaction between E-cadherin and InlA.

Yersinia enterocolitica biovar 1A strains have been delineated into two clonal groups (A and B) based on repetitive extragenic palindrome- and enterobacterial repetitive intergenic consensus-PCR genotyping. The present study investigated the interaction of Y. enterocolitica biovar 1A strains with cultured cells in vitro by their ability to adhere, invade and survive within these cells. The response of macrophages to these strains was also studied by quantifying the expression of inducible nitric oxide synthase, production of nitric oxide and cytokines, and activation of NFκB. The survival rate of clonal group B strains inside macrophages was significantly higher than that of clonal group A strains. In addition, strains harbouring the fepA gene showed better survival inside macrophages. However, the production of nitric oxide and cytokines and activation of NFκB did not show any significant differences between the two clonal groups. In this study, interaction of Y. enterocolitica biovar 1A with cultured cells in vitro did not reflect the previously identified clonal groups, but was more dependent on the characteristics of the individual strains. Therefore, a combination of genotype and phenotype data must be used to characterize this extremely heterogeneous organism.

The aim of this study was to compare the virulence characteristics and phylogenetic features of enteroaggregative Escherichia coli (EAEC) strains from adults with and without diarrhoea and to search for associations between the analysed genes and carrier or diarrhoeagenic strains, respectively. Faecal samples of 487 healthy humans were screened for EAEC strains and compared with isolates from diarrhoeal patients. Virulence and virulence-associated gene typing, serotyping, multilocus sequence typing and antibiotic susceptibility testing were performed for characterization of the isolates. Characteristics significantly linked to carrier strains or to diarrhoeagenic strains were determined. From 487 stool samples, 24 EAEC strains were obtained. Comparison with strains originating from diseased persons showed a statistically significant association of the genes sat (P = 0.002) and agg3C (P = 0.0139) with the carrier strains, and of pCVD432 (P = 0.0001), aap (P = 0.003), aggR (P = 0.0048) and air (P = 0.031) with the diarrhoeagenic strains. Our study indicates that a certain subset of EAEC is unrelated to diarrhoea, for which sat and agg3C may be markers. Our results further suggest that diarrhoeagenic EAEC strains are distinguishable from carrier strains and suggest that, in addition to well-established markers such as pCVD432 and aggR, aap and air may be useful additional markers to define EAEC as an aetiological agent of diarrhoea in adults.

Two assays (Liaison, Diasorin; IDEIA, Oxoid) for detection of Borrelia-specific antibodies were compared. A case–control design using patients with neuroborreliosis (n = 48), laboratory defined by a positive Borrelia-specific antibody index in the spinal fluid, was available and was intended to represent the serological response of disseminated early Lyme borreliosis in general. Serum samples were obtained from 216 Danish blood donors as controls. By comparing sensitivity and specificity using pre-specified cut-off values, significant differences were found. However, using receiver operating characteristic (ROC) curves to optimize and standardize test interpretation, it was shown that testing with both IDEIA IgG and IgM was comparable to testing with Liaison IgG alone by comparing the area under the curve of the diagnostically relevant 25 % partial ROC curve (P = 0.1). When using the Liaison OspC/VlsE IgM assay, the specificity was decreased without a gain in sensitivity. This study proposes standardizing of reporting by using a control population as the reference and choosing decision thresholds guided by the risk of false-positive results at 2 and 8 %. The sensitivities for IDEIA (IgG and IgM combined) were 85 and 95 % and for the Liaison (VlsE IgG) method were 67 and 96 %, respectively. Methods for test evaluation, test interpretation and statistical testing are presented and discussed. In conclusion, Liaison VlsE IgG alone and IDEIA IgG/IgM combined showed a high and comparable discriminatory ability to distinguish serum samples from patients with neuroborreliosis from blood donor controls. However, cut-off values should be adjusted for a proper comparison.

Chlamydia are Gram-negative obligate bacteria that cause a wide range of diseases in humans and animals. To assess the risk of zoonosis posed by pigs, a total of 920 serum samples were collected from pigs in 11 administrative cities in Jiangxi province, south-eastern China, and the seroprevalence of Chlamydia antibodies was investigated by an indirect haemagglutination assay. The pathogen-specific antibodies were detected in 539 (58.59 %) pigs with seroprevalence ranging from 33.33 % (Jingdezhen) to 90.91 % (Pingxiang) among different cities (P<0.05). The highest prevalence was found in pregnant sows (80.89 %, 127/157), followed by breeding boars (79.37 %, 50/63), suckling sows (77.01 %, 67/87), fattening pigs (69.32 %, 61/88) and non-pregnant sows (62.5 %, 180/288). Piglets had the lowest prevalence of 22.78 % (54/237). The seroprevalence of Chlamydia infection among different categories of pigs was also significantly different (P<0.05). These results indicate that Chlamydia is highly prevalent in pigs in Jiangxi province and our results indicate that the presence of Chlamydia exposure in pigs may pose a potential threat to human health.

Antibiotic resistance in Streptococcus pneumoniae is a major concern worldwide. However, it is unclear whether resistance is associated with only a few highly prevalent clones or numerous and diverse clones. We monitored 349 healthy children and obtained nasopharyngeal cultures at five time points coinciding with health check-ups (4, 7, 10, 18 and 36 months) between 2008 and 2012. A total of 497 S. pneumoniae isolates from 257 healthy children were characterized using capsular serotyping, multilocus sequence typing and antibiotic resistance genotyping (ermB, mefA/E and pbp mutations). Among these isolates, 25 serotypes and 66 sequence types (STs) were found, including 24 new STs with 11 new alleles. Although resistance was present in a variety of ST clones, most of the clones (57/66, 86.4 %) had one specific resistant or susceptible genotype. Of 233 phenotypically penicillin-non-susceptible isolates, 196 (84.1 %) belonged to only six clones, comprising ST906B, ST23619F, ST24223F, ST37876A, ST143723F and ST33823A and their variants. We concluded that drug-resistant S. pneumoniae is associated with a limited number of highly prevalent clones that are capable of adapting to the community setting. Furthermore, we analysed the capsular gene evolution in serogroup 6. The strain ST29246D was probably the result of recombination of a 3563 bp fragment of the capsule locus acquired by an ST29246C strain from an ST906B or ST29246B strain. Compared with previous studies, our results showed a different recombination site (wciN and wzx) and a different cps profile (8-7-11), indicating that serogroup 6 strains have multiple sites for cps recombination as a mechanism of vaccine escape.

An oleate-dependent Enterococcus faecalis isolate representing small-colony variants (SCVs) was isolated from the umbilical exudate of a 31-month-old Japanese male patient in Nagano Children’s Hospital, Azumino, Japan. The patient had been suffering from recurrent omphalitis since early infancy. The initial E. faecalis SCV isolate formed small colonies on sheep blood agar plates and tiny colonies on chocolate and modified Drigalski agar, although no visible growth was observed in HK‐semi solid medium after 48 h incubation in ambient air. Moreover, the SCV isolate, the colonial morphology of which was reminiscent of Streptococcus species, could not be identified using the MicroScan WalkAway-40 and API 20 Strep systems, both of which yielded profile numbers that did not correspond to any bacterial species, probably as a result of insufficient growth of the isolate. The SCV isolate was subsequently identified as E. faecalis based on its morphological, cultural and biochemical properties, and this was confirmed by sequencing the 16S rRNA gene of the organism. Investigations revealed that the addition of oleate, an unsaturated fatty acid, enabled the isolate to grow on every medium with normal-sized colony morphology. Although it has long been known that long-chain fatty acids, especially unsaturated oleic acid, have a major inhibitory effect on the growth of a variety of microorganisms, including not only mycobacteria but also streptococci, this is, to the best of our knowledge, the first clinical isolation of an oleate-dependent E. faecalis SCV isolate. In addition, oleic acid might be considered to affect the cell membrane permeability of carbohydrates or antimicrobial agents such as β-lactams.

The aim of this study was to evaluate the effect of heat stress in in vitro conditions on the induction of heat-shock protein (Hsp)70 by Escherichia coli cells, and to determine the localization of Hsps in cell fractions. The material consisted of wild strains of E. coli isolated from the digestive tract of calves, suspended in an exponential-phase culture and subjected to 41.5 °C for 2 h. Individual fractions were analysed by SDS-PAGE and two-dimensional electrophoresis. Western blotting with mouse anti-Hsp70 and anti-Hsp60 mAbs was used to identify the proteins. Electrophoretic analysis of the heat-treated cells detected Hsp70 in all three fractions, cytoplasmic, periplasmic and membrane, which was confirmed by Western blotting. The proteins obtained had diverse localizations in the pH gradient in two-dimensional electrophoresis, which may indicate changes in their conformation and physical properties leading to stabilization and protection of intracellular structures in stress conditions. The presence of these Hsps in different cell fractions indicates a very strong protective adaptation in the bacteria in unfavourable conditions, which is critical for the organism infected by them.

The primary pathogens found in men with urethritis are Chlamydia trachomatis and Neisseria gonorrhoeae. Rapid diagnosis of N. gonorrhoeae infection can be made based on a Gram- or methylene blue-stained urethral smear. We describe a case of a man with purulent penile discharge, in which microscopic examination led to the presumptive diagnosis of gonorrhoea. A nucleic acid amplification test was negative for N. gonorrhoeae but positive for C. trachomatis. Culture showed Gram-negative diplococci which were identified as Neisseria meningitidis. N. meningitidis can be sporadically pathogenic in the genito-urinary tract and mimicks gonococcal urethritis, and appears identical by microscopy. When a gonococcal urethritis is suspected based on clinical signs and microscopic examination, but investigatory tests cannot confirm the diagnosis, a N. meningitidis infection should be considered.

Diffusely adherent Escherichia coli (DAEC) is thought to cause diarrhoea in children, and so too are other diarrhoeagenic E. coli (DEC); however, the evidence base is inconclusive. DEC pathotypes are differentiated on the basis of their pathogenic features, and thus cannot be quickly identified on selective culture media. Molecular techniques, not readily available in most clinical laboratories, are required to differentiate DEC strains from non-pathogenic E. coli in the stool flora. We report a case of persistent bloody diarrhoea, without fever, in a previously healthy 21-month infant from whom we isolated five DAEC strains. The child’s stools movements were loose, with gross blood and mucus; fresh mount analysis revealed numerous faecal leukocytes and erythrocytes. Response to antimicrobial treatment with trimethoprim-sulfamethoxazole was poor despite susceptibility in vitro. Although the patient improved with azithromycin, blood was present in the patient’s stools for over 30 days. The severe diarrhoea in this patient might be explained by the fact that these DAEC isolates harboured a siderophore receptor, which allows the bacteria to use iron derived from haem compounds that promote its multiplication. The isolates also induced in vitro secretion of several immunomodulatory cytokines that may account for the patient’s loose stools and faecal leukocytes. DAEC may play a greater role than suspected in afebrile children with bloody diarrhoea.

Infective endocarditis is a very rare clinical form caused by Erysipelothrix rhusiopathiae. It is rarely seen in immunocompetent individuals. Even after surgery it may entail mortality rates as high as 30–40 %. This report describes a case of native valve endocarditis caused by E. rhusiopathiae and cured with crystallized penicillin G and surgery.