- Volume 65, Issue 11, 2016

In order to develop novel host/pathogen real-time PCR assays for routine diagnostic use, early gene expression patterns from both Epstein–Barr virus (EBV) and Raji cells were examined after inducing the lytic life cycle using 12-O-tetradecanoyl-13-phorbol ester and sodium butyrate. Real-time PCR identified several highly induced (>90-fold) EBV lytic genes over a 48 h time course during the lytic induction phase. Latent genes were induced at low levels during this phase. The cellular response to lytic viral replication is poorly understood. Whole human genome microarray analysis identified 113 cellular genes regulated twofold or more by EBV, including 63 upregulated and 46 downregulated genes, over a 24 h time course post-induction. The most upregulated gene was CHI3L1, a chitinase-3-like 1 protein (18.1-fold; P<0.0084), and the most downregulated gene was TYMS, a thymidylate synthetase (−7.6-fold). Gene Ontology enrichment analysis using MetaCore software revealed cell cycle (core), cell cycle (role of anaphase-promoting complex) in cell cycle regulation) and lymphatic diseases as the most significantly represented biological network processes, canonical pathways and disease biomarkers, respectively. Chemotaxis, DNA damage and inflammation (IL-4 signalling) together with lymphoproliferative disorders and non-Hodgkin’s lymphoma were significantly represented biological processes and disease biomarkers.

New Delhi metallo-β-lactamase (NDM)-producing Enterobacteriaceae (NPE) shows prevalence in China. Little is known about the mechanisms related to the spread of NPE. Recently, a total of 51 non-duplicated NPE isolates were collected from a tertiary-care hospital in China and analysed for genetic relatedness by PFGE, antimicrobial susceptibility by Etest and sequence type by multilocus sequence typing. S1-PFGE and Southern blot analysis or PCR amplification were used for plasmid profiling. Between 2014 and 2015, 22 Escherichia coli, 10 Klebsiella pneumoniae, 9 Enterobacter cloacae, 2 Enterobacter aerogenes, 3 Providencia rettgeri, 1 Klebsiella oxytoca, 1 Proteus mirabilis, 1 Citrobacter freundii, 1 Citrobacter werkmanii and 1 Raoultella planticola were identified as NPE. Results of PFGE and multilocus sequence typing showed that most strains were genetically unrelated. Among the 45 bla NDM-carrying plasmids, there were 25 IncX3 plasmids with a size of about 30 to 50 kb, one 100 kb IncX3 plasmid, 11 IncA/C plasmids with a size range from 70 to 300 kb, six 90 to 120 kb IncB/O plasmids, one IncN plasmid with a size of 100 kb and one 140 kb IncFrep plasmid. An NDM-1-producing isolate of C. werkmanii was identified, which had not been reported previously. An Escherichia coli strain was found acquiring a bla NDM-1-carrying IncFrep plasmid in vivo during infection. In conclusion, an NDM-1-producing isolate of C. werkmanii was identified. An Escherichia coli strain acquired a bla NDM-1-carrying plasmid in vivo. IncX3 and IncA/C plasmids with various sizes might have emerged as the main platforms mediating the spread of the bla NDM genes in China.

Two clinical isolates from Polish patients, Proteus mirabilis 9B-m and Proteus genomospecies 3J-r, were found to be serologically related to P mirabilis O11. However, serological studies involving ELISA and Western blotting methods, using lipopolysaccharides (LPSs) extracted from the strains as antigens and native or adsorbed rabbit polyclonal O antisera, specific to the studied strains, revealed slight differences in the cross-reactivity and specificity of the two studied Proteus isolates, when compared to P. mirabilis O11. Two different O polysaccharides containing N-(d-galacturonoyl)-l-threonine were isolated from the LPSs of the isolates. Their structures were determined by chemical analysis and NMR spectroscopy and found to be related to the P. mirabilis O11 antigen structure established earlier, the 9B-m structure differing in the absence of the lateral glucose residue and the 3J-r structure in non-stoichiometric O-acetylation of the threonine residue only. Thus, the Proteus O11 serogroup should be divided into two subgroups: O11a, represented by the 9B-m isolate and O11a, b possessing the additional b epitope, containing the lateral residue of glucose and formed by the 3J-r isolate as well as P. mirabilis 25/57 belonging to O11 serogroup so far. O11a is the sixth new serotype found in Proteus spp. strains recently isolated from patients in central Poland.

Genetic mechanisms of methicillin resistance are still relevant in staphylococci. The aims of this study are to assess the possible exchanges of staphylococcal cassette chromosome mec (SCCmec) among isolates of methicillin-resistant staphylococci (MRS) and to check for known or new mutations in mecA DNA. A total of 35 MRS non-repetitive isolates were recovered, including 20 Staphylococcus haemolyticus, 7 Staphylococcus aureus, 4 Staphylococcus sciuri, 2 Staphylococcus saprophyticus and 1 isolate each of Staphylococcus xylosus and Staphylococcus lentus. Only 16 of the 35 strains were assigned to known SCCmec types: 7 SCCmec VII, 6 SCCmec IV and 3 SCCmec III, with possible horizontal transfer of the SCCmec VII from methicillin-resistant S. haemolyticus to methicillin-susceptible S. aureus. mecA gene sequencing in ten selected isolates allowed description of nine punctual mutations, seven of which were reported for the first time. The most frequent mutation was G246E, identified in isolates of methicillin-resistant S. aureus, S. sciuri, S. saprophyticus and S. lentus. These results emphasized the high degree of genetic diversity of SCCmec element in MRS and describe new missense mutations in mecA, which might be important in understanding the evolution of methicillin and new β-lactam resistance.

During the 2014/15 winter season, a newly emergent GII.P17-GII.17 variant overwhelmed currently dominant GII.4 viruses, causing outbreaks of acute gastroenteritis in China and Japan. In Nanjing area, this novel GII.17 variant was first identified in a sporadic case of acute gastroenteritis in July 2013, 18 months ahead of reports from other parts of China. In this study, epidemiological features and genotyping of noroviruses from 2013 to 2015 were depicted. Twenty-eight local GII.17 sequences of capsid N-terminus originating from July 2013 to August 2015 were aligned, among which complete genome of seven strains obtained from two outbreaks and five sporadic cases was extensively characterized. The differences of local GII.17 variants led to at least two clusters, with strains from 2013/14 season and those from 2014/15 season grouped differently. Multiple nucleotide and amino acid variations between different clusters of GII.17 were elucidated, including residue substitutions and insertion occurring in or near antigenic and receptor-binding sites of viral protein 1. In addition, sequence hypervariability from residue 279 through 406 of viral protein 2 was identified. The modifications may reveal a distinctive adaptive process which could in part explain the rapid spread of emerging GII.17 variants. Continued monitoring on novel GII.17 is essential.

Burkholderia pseudomallei, the causative agent of melioidosis, has been found to increase its resistance to antibiotics when growing as a biofilm. The resistance is related to several mechanisms. One of the possible mechanisms is the efflux pump. Using bioinformatics analysis, it was found that BPSL1661, BPSL1664 and BPSL1665 were orthologous genes of the efflux transporter encoding genes for biofilm-related antibiotic resistance, PA1874–PA1877 genes in Pseudomonas aeruginosa strain PAO1. Expression of selected encoding genes for the efflux transporter system during biofilm formation were investigated. Real-time reverse transcriptase PCR expression of amrB, cytoplasmic membrane protein of AmrAB-OprA efflux transporter encoding gene, was slightly increased, while BPSL1665 was significantly increased during growth of bacteria in biofilm formation. Minimum biofilm inhibition concentration and minimum biofilm eradication concentration (MBEC) of ceftazidime (CTZ), doxycycline (DOX) and imipenem were found to be 2- to 1024-times increased when compared to their MICs for of planktonic cells. Inhibition of the efflux transporter by adding phenylalanine arginine β-napthylamide (PAβN), a universal efflux inhibitor, decreased 2 to 16 times as much as MBEC in B. pseudomallei biofilms with CTZ and DOX. When the intracellular accumulation of antibiotics was tested to reveal the pump inhibition, only the concentrations of CTZ and DOX increased in PAβN treated biofilm. Taken together, these results indicated that BPSL1665, a putative precursor of the efflux pump gene, might be related to the adaptation of B. pseudomallei in biofilm conditions. Inhibition of efflux pumps may lead to a decrease of resistance to CTZ and DOX in biofilm cells.

Mycobacterium chimaera arouses an increasing public health concern, as this non-tuberculous mycobacterium (NTM) has recently been associated with life-threatening cardiac infections. M. chimaera and Mycobacterium intracellulare are genetically very close but recently appeared to present different epidemiological and clinical significance. Therefore, it has become important for laboratories to use adequate techniques allowing precise species identification. To date, most commercially available laboratory assays cannot distinguish them and erroneously identify M. chimaera as M. intracellulare. We performed a re-analysis of the 149 M. intracellulare strains received by the Belgian National Reference Laboratory using 16S rRNA gene sequencing, representing 25 % of all NTM collected in 2015. We found that M. chimaera represents the majority (n=94, 63 %) of the previous M. intracellulare. This study reports the large presence of M. intracellulare/chimaera among Belgian patients infected by an NTM and the predominance of the species M. chimaera among this group. This study also stresses the public health importance of M. chimaera and demonstrates the inability of commonly used laboratory techniques to correctly diagnose these infections.

Corynebacterium diphtheriae is typically recognized as a colonizer of the upper respiratory tract (respiratory diphtheria) and the skin (cutaneous diphtheria). However, different strains of Corynebacterium diphtheriae can also cause invasive infections. In this study, the characterization of a non-toxigenic Corynebacterium diphtheriae strain (designated BR-INCA5015) isolated from osteomyelitis in the frontal bone of a patient with adenoid cystic carcinoma was performed. Pathogenic properties of the strain BR-INCA5015 were tested in a Caenorhabditis elegans survival assay showing strong colonization and killing by this strain. Survival rates of 3.8±2.7 %, 33.6±7.3 % and 0 % were observed for strains ATCC 27010T, ATCC 27012 and BR-INCA5015, respectively, at day 7. BR-INCA5015 was able to colonize epithelial cells, showing elevated capacity to adhere to and survive within HeLa cells compared to other Corynebacterium diphtheriae isolates. Intracellular survival in macrophages (THP-1 and RAW 264.7) was significantly higher compared to control strains ATCC 27010T (non-toxigenic) and ATCC 27012 (toxigenic). Furthermore, the ability of BR-INCA5015 to induce osteomyelitis was confirmed by in vivo assay using Swiss Webster mice.