- Volume 59, Issue 4, 2010

Congenital sensorineural hearing loss (SNHL) is common. In theWestern world, the incidence is 1–3 per 1000 live births. The aetiologyencompasses genetic and non-genetic factors accounting for 55 %and 45 % of cases, respectively. Reports that describe the contributionof intrauterine infection to the occurrence of congenital SNHL are limited,and comparative analysis of the different pathogens is lacking. Lipopolysaccharide (LPS),a product of bacteriolysis, has been demonstrated to be associated with innerear damage in experimental studies. To elucidate the potential role of thistoxin in congenital SNHL and to identify the pathogenesis and transmissionroutes, we reviewed the literature. We speculate that different routes ofexposure to LPS in utero may result in congenital inner ear damage.

Continuous subculture has been observed to produce changes in the virulenceof micro-organisms, e.g. rabies virus, poliovirus and Mycobacterium bovis BCG. The latter has been used as a vaccine for tuberculosis for thelast 100 years; however, in some instances its efficacy has been observedto be very low. In order to determine whether similar changes can be producedin Mycobacterium tuberculosis, we selected four isolates, M.tuberculosis H37Rv, a Beijing strain (DR-689), and two moreisolates with deletion of the phospholipase C locus (plcA-plcB-plcC), and subjected them to serial culturing on Middlebrook 7H9 medium,with or without ox bile. After 100 passages, we performed RFLP-IS6110 analysis to determine whether genomic changes were produced. We alsochecked their genomic composition by microarray analysis. Changes in virulencewere studied by measuring the cytotoxic effect of parental and subculturedisolates on a THP-1 macrophage monolayer. The most visible change was thechange of position of an IS6110 band of ∼1400 bp to ∼1600 bpin the Beijing isolate subcultured in the ox bile medium. Analysis by microarrayand PCR confirmation did not reveal any genomic changes. Cytotoxic activitywas decreased in the isolates at levels close to that of BCG, and more consistentlyin those subcultured in the presence of ox bile.

Many bacterial infections are associated with biofilm formation. Bacterialbiofilms can develop on essentially all kinds of surfaces, producing chronicand often intractable infections. Escherichia coli is an importantpathogen causing a wide range of gastrointestinal infections. E. coli strain Nissle 1917 has been used for many decades as a probiotic againsta variety of intestinal disorders and is probably the best field-tested E. coli strain in the world. Here we have investigated the biofilm-formingcapacity of Nissle 1917. We found that the strain was a good biofilm former.Not only was it significantly better at biofilm formation than enteropathogenic,enterotoxigenic and enterohaemorrhagic E. coli strains, it was alsoable to outcompete such strains during biofilm formation. The results supportthe notion of bacterial prophylaxis employing Nissle 1917 and may partiallyexplain why the strain has a beneficial effect on many intestinal disorders.

Leprosy, a spectral disease manifested on the basis of host immune responses,is complicated by its reactional stages, namely type I reversal reaction (RR)and type II erythema nodosum leprosum (ENL). These reactional stagesare characterized by uncontrolled and aberrant immune responses. Biomarkersfor reactional stages would aid in early diagnosis, efficient treatment, preventionof neurological complications and prediction of predisposition to reactionalstages. In this study, comparative analysis of the serum proteome of leprosypatients by two-dimensional electrophoresis (2DE) followed by massspectrometry showed differential expression of acute-phase protein α 1-acid glycoprotein (AGP; also known as orosomucoid).AGP levels in untreated ENL cases were significantly higher than in lepromatousleprosy (LL; a non-reactional disease stage) (P=0.0126),RR (P=0.0176) and healthy controls (P=0.0030).These data were confirmed using ELISA. The levels of AGP decreased to normallevels after treatment with multidrug therapy and thalidomide (P=0.0167). In a follow-up study, AGP levels, which were highin the untreated ENL stage, decreased significantly at 5 days (P=0.0084) and 21 days (P=0.0027)post-treatment. A stage-dependent increase in AGP in an LL patient who progressedinto the ENL stage was also shown. Glycosylation analysis by 2DE showed differentialexpression of acidic glycoforms of AGP in untreated ENL cases. Changes inAGP concentration and differential expression of isoforms correlated withthe inflammatory condition in ENL and also with the treatment regimen. Thus,initial validation of AGP as an ENL-specific biomarker and treatment indicatorwas shown in this study.

Aspergillus fumigatus is the major cause of invasive aspergillosis (IA),a disease associated with high rates of morbidity and mortality in patientsundergoing treatment for haematological malignancies. This study investigated A. fumigatus growth in vitro and in a murine model of IA inorder to provide insights into the dynamics of extracellular DNA and galactomannan (GM)release and their relevance to early diagnosis of IA. Following inoculationof whole blood with 20 A. fumigatus conidia ml−1,DNA that corresponded to the inoculum could be detected by PCR but GM wasnot detected in plasma separated from the blood sample, indicating that thefungus did not grow in whole blood. The quantities of DNA detected by PCR,and GM, were proportional to the amount of fungal biomass present in vitro. Fungal DNA could be detected in the sera of mice experimentally infectedwith A. fumigatus with maximum detection in cyclophosphamide-treatedmice.

Candida orthopsilosis and Candida metapsilosis are recentlydescribed species phenotypically indistinguishable from Candida parapsilosis. We evaluated phenotyping and molecular methods for the detection ofthese species among 79 unique blood culture isolates of the C. parapsilosis group obtained during the years 2004–2008. The isolates were screenedby PCR amplification of the secondary alcohol dehydrogenase-encoding gene (SADH) followed by digestion with the restriction enzyme BanI, using C. parapsilosis ATCC 22019, C. orthopsilosisATCC 96139 and C. metapsilosis ATCC 96144 as controls. Isolates withRFLP patterns distinct from C. parapsilosis were characterized bysequence analysis of the ITS1–ITS2, 26S rRNA (D1/D2) and SADH regions. Restriction patterns for the 3 species with each of 610restriction enzymes were predicted in silico using 12 available sequences.By PCR-RFLP of the SADH gene alone, four isolates (5.1 %)had a pattern identical to the C. orthopsilosis reference strain.Sequence analysis of SADH and ITS (internal transcribed spacer)regions identified two of these isolates as C. metapsilosis. Theseresults were confirmed by creating a phylogenetic tree based on concatenatedsequences of SADH, ITS and 26S rRNA gene sequence regions. Optimaldifferentiation between C. parapsilosis, C. metapsilosisand C. orthopsilosis was predicted using digestion with NlaIII,producing discriminatory band sizes of: 131 and 505 bp; 74, 288 and 348 bp;and 131, 217 and 288 bp, respectively. This was confirmed using the referencestrains and 79 clinical isolates. In conclusion, reliable discrimination wasobtained by PCR-RFLP profile analysis of the SADH gene after digestionwith NlaIII but not with BanI. C. metapsilosisand C. orthopsilosis are involved in a small but significant numberof invasive infections in Denmark.

Brucellosis is a disease caused by Gram-negative, facultative, intracellularbacteria belonging to the genus Brucella. It is an emerging zoonosis,and an economically important infection of humans and livestock with a worldwidedistribution. Human infection is known to occur through consumption of infectedraw milk, milk products and undercooked or raw meat. Serodiagnosis of brucellosisis carried out by detection of antibodies generated against LPS or whole-cellbacterial extracts by ELISA or agglutination tests using colorimetry. Thepresent study was designed to develop a highly sensitive and specific indirectELISA in both a microtitre plate and dot-blot format employing the recombinantouter-membrane protein 28 (rOmp28). Cloning and expression of Brucella melitensis Omp28 protein, which is a group 3 antigen, was accomplishedby PCR amplification and cloning of the gene in a pET-28a expression system,followed by Ni-NTA affinity chromatography purification of the His-taggedrecombinant protein. An indirect ELISA in both a microtitre plate and dot-blotformat was optimized with sera collected from three groups: culture-confirmedcases, clinically suspected cases and healthy individuals. The rOmp28 proteinreacted only with the culture-confirmed positive samples and no reaction wasobserved with culture-negative samples, confirming the immunoreactivity ofthe recombinant protein. The test in both formats had a correlation of approximately90 % with the Rose Bengal plate agglutination test (RBPT)and a standard tube agglutination test, assays that are routinely performedfor the serodiagnosis of brucellosis. The sensitivity and specificity of theassay in the plate format were 97.50 and 85.59 %, and in thedot-blot format were 82.05 and 92.43%, respectively, in comparisonwith RBPT. The specificity of this assay was further confirmed by testingsamples that were positive for malaria and typhoid, which gave negative results.This ELISA system in microtitre plates and a dot-blot format will be usefulfor the rapid screening of large numbers of samples for the diagnosis of humanbrucellosis in endemic areas.

Acute lung injuries due to acute lung infections remain a major cause ofmortality. Thus a combination of an antibiotic and a compound with immunomodulatoryand anti-inflammatory activities can help to overcome acute lung infection-inducedinjuries. Curcumin derived from the rhizome of turmeric has been used fordecades and it exhibits anti-inflammatory, anti-carcinogenic, immunomodulatoryproperties by downregulation of various inflammatory mediators. Keeping theseproperties in mind, we investigated the anti-inflammatory properties of curcuminin a mouse model of acute inflammation by introducing Klebsiella pneumoniae B5055 into BALB/c mice via the intranasal route. Intranasal instillationof bacteria in this mouse model of acute pneumonia-induced inflammation resultedin a significant increase in neutrophil infiltration in the lungs along withincreased production of various inflammatory mediators [i.e. malondialdehyde (MDA),myeloperoxidase (MPO), nitric oxide (NO), tumour necrosisfactor (TNF)-α] in the lung tissue. The animalsthat received curcumin alone orally or in combination with augmentin, 15 daysprior to bacterial instillation into the lungs via the intranasal route, showeda significant (P <0.05) decrease in neutrophil influxinto the lungs and a significant (P <0.05) decreasein the production of MDA, NO, MPO activity and TNF-α levels.Augmentin treatment alone did not decrease the MDA, MPO, NO and TNF-α levels significantly (P >0.05) as compared tothe control group. We therefore conclude that curcumin ameliorates lung inflammationinduced by K. pneumoniae B5055 without significantly (P <0.05) decreasing the bacterial load in the lung tissue whereasaugmentin takes care of bacterial proliferation. Hence, curcumin can be usedas an adjunct therapy along with antibiotics as an anti-inflammatory or animmunomodulatory agent in the case of acute lung infection.

The postantibiotic effect (PAE) of tomopenem was determined aftera 2 h exposure of two strains of meticillin-susceptible and meticillin-resistant Staphylococcus aureus (MSSA and MRSA), and imipenem-susceptibleand imipenem-resistant Pseudomonas aeruginosa, to tenfold the respectiveMIC. The PAEs on MSSA and P. aeruginosa were approximately 1 hand they were comparable to those of meropenem. The PAE on MRSA was 1.5 to3 h, equal to or longer than those of vancomycin. The PAEs of tomopenemnot only were found for MRSA, but also were present in the imipenem-resistant P. aeruginosa tested.

Streptococcus pyogenes is an important human pathogen for whichan association between infection site and selected epidemiological or functionalmarkers has previously been suggested. However, the studies involved oftenused strains with an insufficiently defined clinical background and laboratoryhistory. Thus, the major goal of the present study was to investigate theserelationships in 183 prospectively collected, well-defined, low-passage isolatesfrom a North-East German centre for tertiary care. For each isolate the clinicalbackground (91 respiratory, 71 skin and 21 invasive isolates) andantibiotic-resistance pattern was recorded. All isolates were classified accordingto their emm type, antibiotic-resistance and PFGE pattern (SmaI restriction analysis of genomic DNA). As novel discriminatorymethods we performed a PCR-based typing of the pilus-protein-encoding FCTregion (FCT) and biofilm-formation phenotyping in various culturemedia. Forty-one isolates were found to be resistant to at least one of thetested antibiotics. emm typing revealed emm28, emm12, emm1, emm4, emm89 and emm2 as themost frequent types in our collection. The novel FCT typing showed isolatesencoding FCT types 4 and 2 to be the most common. Overall 113 strains withunique combinations of emm and FCT types, antibiotic-resistance andPFGE patterns were identified. The majority of all isolates revealed an associationof biofilm-formation capacity with growth media. Comparing all results forpotential associations, no correlation could be established between the anatomicalsite of isolation and the emm or the FCT type. There was no relationshipbetween biofilm formation and emm type, antibiotic-resistance orPFGE patterns. However, a novel association between biofilm formation andFCT type became obvious among strains from our collection.

Studies based on the analysis of housekeeping genes indicate that Escherichia coli and all Shigella species, except for Shigellaboydii type 13, belong to a single species. This study analysed the phenotypicand genotypic characteristics of 23 E. coli strains isolatedin different countries from faecal specimens taken from children with diarrhoea.Strains were identified using the VITEK system and typed with rabbit seraobtained against 186 somatic and 53 flagellar E. coli antigens andagainst 45 Shigella somatic antigens. Biochemical analysis of thesestrains showed a typical E. coli profile with a defined reactionagainst both E. coli O179 and S. boydii16 somatic antisera. Agglutination assays for flagellar antigens showed aresponse against H2 in 7 (30 %) strains, H10 in 2 (9 %)strains, H32 in 12 (52 %) strains and H34 in 2 (9 %)strains, demonstrating 4 serotypes associated with this new somatic antigen64474. A serum against one of these E. coli strains (64474)was prepared. Absorption assays of S. boydii 16 and E. coli 64474 antisera with E. coli O179 antigen removedthe agglutination response against this O179 antigen completely, while theagglutination titres against both S. boydii 16 and E. coli 64474 remained the same. Four (17 %) E. coli strains showed antimicrobial resistance to piperacillinonly, one (4 %) to piperacillin and trimethoprim/sulfamethoxazole,one (4 %) to ciprofloxacin, nitrofurantoin and piperacillin,and two (9 %) strains were resistant to ciprofloxacin,norfloxacin, ofloxacin, piperacillin and trimethoprim/sulfamethoxazole.With regards to PCR assays, one (4 %) of the strainswas positive for Shigella gene ipaH, one (4 %)for ipaA, two (9 %) for ipaB, one (4 %)for ipaD, two (9 %) for sepA andthree (13 %) for ospF. The rfb genecluster in the E. coli strains was analysed by RFLP and comparedwith the gene cluster obtained from S. boydii 16. The rfb-RFLPpatterns for all 23 E. coli strains were similar to thoseobtained for S. boydii 16. The results from PCR tests todetect rfb genes wzx (encoding O unit flippase)and wzy (encoding polymerase) belonging to a cluster relatedto the biosynthesis of the S. boydii 16-specific O antigen were positivein 21 (91 %) and 22 (96 %)of the strains, respectively. PCR assays to detect E. colivirulence genes were also performed. These assays detected enterotoxigenic E. coli genes ltA1 in 12 of the strains (52 %), st1a in 4 (17 %), cfa1 in 6 (26 %), cs1 in 1 (4 %), cs3 in 3 (13 %), cs13 in 9 (39 %) and cs14 in 5 (22 %)of the strains. Results from the PFGE analyses confirmed the wide geographicaldistribution of these strains suggesting that 64474 : H2, 64474 : H10,64474 : H32 and 64474 : H34 are new serotypesof E. coli strains with a defined virulence capacity, andshare a common O antigen with S. boydii 16.

Acanthamoeba keratitis (AK) is a sight-threatening cornealinfection, the epidemiology of which is related to the specific genotype of Acanthamoeba. In this study, the genotypes of 14 Acanthamoebaisolates, each from a patient with AK, were identified according to the highlyvariable DF3 region in the 18S rRNA gene at Shandong Eye Institute, PR China,from 2000 to 2009, and the clinical characteristics of these patients wereanalysed. All 14 amoebae were genotype T4, representing nine different DF3sequence types, seven of which were newly identified. Cornea infestation wasthe main risk factor for these 14 AK patients. Amoebic cysts could be detectedin all corneal scrapes. Corneal ulcers were located mainly at the cornealcentre, accompanied by eye pain, and some appeared with a Wessely ring. Surgerywas carried out on all patients. Acanthamoeba genotypes T4/26and T4/27 were found to cause a more severe keratitis, whilst the othersshowed no significant differences in clinical characteristics. In conclusion,the majority of the keratitis-causing Acanthamoeba isolates weregenotype T4, with Acanthamoeba genotypes T4/26 and T4/27from PR China causing a more severe keratitis.

This study was designed to determine the genetic basis of florfenicol andceftiofur resistance in Escherichia coli isolates recovered fromFrench cattle. In these isolates, ceftiofur resistance was conferred by bla CMY-2 located on three distinct conjugative plasmids ona specific DNA fragment, ISEcp1-bla CMY-2-blc-sugE. Two of the plasmids also carried the floR gene conferringresistance to florfenicol. The floR gene was shown to be associatedwith the insertion sequence ISCR2. Mobile elements appear to contributeto the mobilization of floR and bla CMY-2 genesin E. coli. The presence of bla CMY-2 and floR on the same plasmid highlights the potential risk for a co-selectionof the bla CMY-2 gene through the use of florfenicol infood animal production.

Chronic respiratory infection by Pseudomonas aeruginosa contributessignificantly to the morbidity and mortality associated with cystic fibrosis (CF).Using a series of phenotypic and genotypic tests on collections of 40 isolatesper sputum sample, we analysed fluctuations within sputum populations of the P. aeruginosa Liverpool epidemic strain (LES) during pulmonaryexacerbations. For each of three patients, three sequential sputum sampleswere analysed: (1) on presentation with exacerbation at the RegionalAdult Cystic Fibrosis Unit, Liverpool; (2) a few days into intravenousantibiotic treatment; (3) when the patient had recovered. Fluctuationswere observed in morphotype distribution, the production of virulence-associatedquorum-sensing-dependent exoproducts (the phenazine compound pyocyaninand the elastase LasA), antibiotic susceptibility profiles and levelsof auxotrophy. PCR assays were used to screen isolates for the presence ofnovel regions of the LES genome (islands and prophages) and to detectfree phages. In one patient there was an increase in the prevalence of theLESGI-5 genomic island during the sampling period from 10 to 97.5 %carriage. LES phages 2–4 were detected in either the majority or allsputum samples tested, indicating widespread phage activity during the samplingperiod. The results of this study are indicative that significant fluctuationsoccur within P. aeruginosa populations during short periods of pulmonaryexacerbation and intravenous antibiotic therapy.

Daptomycin is a novel lipopeptide with activity against Gram-positive organismsincluding enterococci. It is licensed for the treatment of Staphylococcusaureus bacteraemia and right-sided endocarditis, but not endocarditisdue to Enterococcus spp. We report a case of enterococcal prostheticvalve endocarditis with an aortic root abscess in an elderly patient who wasnot fit for surgery. The patient's endocarditis relapsed 9 weeks aftera 6 week course of daptomycin.

Two cases involving polymicrobial culture-negative samples were investigatedby 16S rRNA gene sequencing, with analysis of mixed chromatograms. Fusobacteriumnecrophorum, Prevotella intermedia and Streptococcus constellatus were identified from pleural fluid in a patient with Lemierre'ssyndrome and Neisseria meningitidis and Escherichia coliwere identified from a petechia in a patient with meningococcal disease.

Outbreaks or clusters of community-acquired meticillin-resistant Staphylococcusaureus (CA-MRSA) within families have been reported. We describea family cluster of CA-MRSA skin and soft-tissue infection where CA-MRSA wassuspected because of recurrent infections which failed to respond to flucloxacillin.While the prevalence of CA-MRSA is low worldwide, CA-MRSA should be consideredin certain circumstances depending on clinical presentation and risk assessment.Surveillance cultures of family contacts of patients with MRSA should be consideredto help establish the prevalence of CA-MRSA and to inform the optimal choiceof empiric antibiotic treatment.

Carnobacterium species have been isolated from the environmentand are not regarded as human pathogens, although they are known to causedisease in fish. Only two reports describing isolation of Carnobacterium species from human pus were found in the literature. We report whatwe believe to be the first isolation of Carnobacterium sp. from ahuman blood culture.