Rapid detection of the methicillin-resistance gene, mecA, in coagulase-negative Staphylococci.

Phenotypical methods are routinely used to detect methicillin resistance in Staphylococci. These methods are time-consuming and there are difficulties in detecting all resistant strains carrying the mecA gene. We detected methicillin-resistant Staphylococci in biological samples by PCR amplification of mecA, without the time-consuming step of identifying a bacterial isolate. Methicillin-resistant Staphylococci isolates were also detected by screening on agar supplemented with oxacillin. The biological samples were collected from the hands of 17 healthcare workers at the Department of Paediatrics at the University Hospital of Tromsø. mecA was amplified in 12 of the 17 samples. The gene was verified by DNA sequencing of the PCR amplicon. Using the phenotypical method, methicillin-resistant Staphylococci were isolated from 6 of the samples. In all 6 of these samples, mecA was amplified by PCR. We conclude that PCR is a sensitive and specific method for detecting methicillin resistance in Staphylococci. The PCR detection of mecA is rapid, fairly simple and can easily be assimilated into the routines of a clinical microbiological laboratory.

Lactoferricin B causes depolarization of the cytoplasmic membrane of Escherichia coli ATCC 25922 and fusion of negatively charged liposomes.

Antimicrobial peptides have been extensively studied in order to elucidate their mode of action. Most of these peptides have been shown to exert a bactericidal effect on the cytoplasmic membrane of bacteria. Lactoferricin is an antimicrobial peptide with a net positive charge and an amphipatic structure. In this study we examine the effect of bovine lactoferricin (lactoferricin B; Lfcin B) on bacterial membranes. We show that Lfcin B neither lyses bacteria, nor causes a major leakage from liposomes. Lfcin B depolarizes the membrane of susceptible bacteria, and induces fusion of negatively charged liposomes. Hence, Lfcin B may have additional targets responsible for the antibacterial effect.

Continuing high prevalence of VanA-type vancomycin-resistant enterococci on Norwegian poultry farms three years after avoparcin was banned.

Avoparcin was used as a feed additive in Norwegian broiler and turkey production from 1986 until 1995. It was banned due to the selection of VanA-type vancomycin-resistant enterococci (VRE) in animal husbandry and to reduce the potential for human exposure to VRE. The aim of the present study was to investigate the prevalence of VRE carriage in Norwegian poultry farmers and their poultry three years after avoparcin was banned. Corresponding faecal samples from poultry and humans on farms where avoparcin had previously been used (exposed farms, n = 73) and farms where avoparcin had never been used (unexposed farms, n = 74) were analysed for the presence of VRE. For each farm, one sample from the poultry house and one sample from the farmer were obtained. VRE were isolated from 72 of 73 (99%) and eight of 74 (11%) poultry samples from exposed and unexposed farms, respectively. VRE were isolated from 13 of 73 (18%) and one of 74 (1%) farmer samples from exposed and unexposed farms, respectively. All VRE isolates were highly resistant to vancomycin and possessed the vanA gene, as shown by PCR. The high prevalence of VRE is in accordance with previous Norwegian studies, and shows a remarkable stability of the VanA resistance determinant in an apparently non-selective environment.

Typeability of Tn1546-like elements in vancomycin-resistant enterococci using long-range PCRs and specific analysis of polymorphic regions.

Molecular subtyping of the VanA-type resistance element Tn1546 in an international collection of 81 genomically diverse vancomycin-resistant enterococci (VRE) from human, animal, and environmental reservoirs was evaluated by restriction analysis of long-range PCR amplicons (PCR-RFLP), single gene PCRs, Southern blot analysis of genomic digests, and partial DNA sequencing. A dominant Tn1546-RFLP in accordance with Enterococcus faecium BM4147 was detected in 43 of the 49 long-range PCR positive strains from ecologically diverse sources in several European countries and the US. Tn1546-like elements from the 32 (40%) long-range PCR negative strains were typed into 17 different groups by single-gene PCRs and Southern blot analysis of the ORF1, ORF2, vanS-vanH, vanX-vanY, and vanZ regions. All these isolates showed deletions in the ORF1 and/or vanZ primer binding regions explaining the failure of long-range PCR amplification. Enlarged vanS-vanH or vanX-vanY fragments were detected in 7 (22%) and 16 (50%) of the long-range PCR negative strains, respectively. The enlarged vanS-vanH regions of five clinical isolates from the US (n = 2), Ireland (n = 2), and Norway (n = 1) contained identical IS1251-like insertions indicating intercontinental spread of the vanA gene cluster. Intergenic vanS-vanH IS1251 insertions have so far not been reported in European studies. Structural rearrangements of Tn1546-like elements may represent single recombination events that can serve as fingerprints in the molecular examination of vanA gene cluster evolution and transmission. The optimal strategy for such analysis has yet to be determined. Two alternative long-range PCRs with subsequent RFLP analysis were successfully used to type the majority of vanA gene clusters in an ecologically and geographically heterogeneous VRE strain collection, but failed to detect and type a group of variant Tn1546-like elements truncated in the left-end ORF1/ORF2 region. Further subtyping of such variants should specifically target the polymorphic vanS-vanH and vanX-vanY regions.

[Antibiotic treatment of acute otitis in children]. / Antibiotikabehandling av akutt ørebetennelse hos barn.

Treatment of acute otitis is one of the most common reasons for prescribing antibiotics for children. Recent studies have shown no, or only a small, effect of antibiotic treatment of this condition. We examined the files from the City of Tromsø's Emergency Department, which catchment area includes about 12,300 children under the age of 15. During the period from March 1997 to May 1998, 784 children presented with acute otitis, 91.5% of whom received antibiotic treatment. The most frequently drug used drug was penicillin V. Even if there are no or only a small effect of antibiotic treatment of this condition, most children receive such treatment. This study will give baseline data for new studies aimed at reducing the use of antibiotics.

Comparison of thymidine kinase and A-type inclusion protein gene sequences from Norwegian and Swedish cowpox virus isolates.

During the last decades, cowpox virus, a member of the genus Orthopoxvirus within the Poxviridae family, has appeared as a pathogen in domestic cats, zoo animal species, and humans. At the same time, vaccinia virus, another orthopoxvirus, has been used as a recombinant vaccine vector with foreign genes inserted in the thymidine kinase (TK) gene. By PCR and cycle sequencing, we have determined the nucleotide sequences of the TK gene and the A-type inclusion protein (ATIP) gene of virus isolates from two human cowpox cases in Sweden, as well as a human and a feline case from Norway. We also obtained the corresponding sequences from ectromelia virus (strain Moscow), cowpox virus (strain Brighton) and vaccinia virus (strain Western Reserve). The new virus isolates differed from ectromelia virus and vaccinia virus, and were confirmed to be cowpox virus strains. Isolates originating from the same country had nearly identical TK sequences and fully identical ATIP sequences. They probably represent local geographical strains of cowpox virus.

Heterogeneity in the vanB gene cluster of genomically diverse clinical strains of vancomycin-resistant enterococci.

Molecular analysis of 17 genomically unrelated clinical VanB-type vancomycin-resistant enterococcus isolates from hospital patients in Germany, Norway, Sweden, the United Kingdom and the United States revealed three subtypes of the vanB gene cluster-vanB1, vanB2, and vanB3-which was in accordance with previous subtyping of the ligase gene sequence. There was no correlation between vanB subtype and levels of vancomycin resistance. All strains studied carried a structurally conserved vanB gene cluster as shown by long-range PCR (long PCR) covering 5,959 bp of the published sequence in vanB1 strain V583. Restriction analysis of long PCR amplicons displayed one unique vanB1 pattern and a second vanB2- and vanB3-specific pattern. The vanSB-vanYB intergenic sequences with flanking coding regions were identical within each vanB subtype with one exception. A U.S. vanB2 isolate had a 789-bp enlargement of this region containing a putative open reading frame (ORF) with substantial homology to an ORF in the Clostridium perfringens IS1469 insertion element. The molecular heterogeneity within the vanB gene cluster has implications for the selection of PCR primers, as the primers must ensure detection of all vanB subtypes, and is of importance when considering reservoirs and dissemination of vanB resistance. The molecular identity within the vanB1 and the vanB2 subtype indicates horizontal transmission of both gene clusters between isolates in different geographical areas. Restriction analysis of long PCR vanB amplicons may reveal specific varieties that can be used as epidemiological markers for mobile determinants conferring VanB-type resistance. The finding of three distinct vanB gene clusters should encourage a search for different environmental reservoirs of vanB resistance determinants.

Detection of a streptomycin/spectinomycin adenylyltransferase gene (aadA) in Enterococcus faecalis.

Genes encoding streptomycin/spectinomycin adenylyltransferases [ANT(3")(9)] have been reported to exist in gram-negative organisms and Staphylococcus aureus. During a study of high-level aminoglycoside resistance in enterococci, we encountered an isolate of Enterococcus faecalis that was streptomycin resistant but did not appear to contain the 6'-adenylyltransferase gene (aadE) when examined by PCR with specific primers. Phosphocellulose paper binding assays indicated the presence of an ANT(3")(9) enzyme. Streptomycin and spectinomycin MICs of 4,000 and 8,000 microg/ml, respectively, were observed for the isolate. PCR primers corresponding to a highly conserved region of the aadA gene were used to amplify a specific 284-bp product. The product hybridized with a digoxigenin-labeled PCR product from E. coli C600(pHP45Omega) known to contain the aadA gene. The aadA gene was transferred via filter matings from the E. faecalis donor to E. faecalis JH2-2. PCR primers designed for analysis of integrons were used to amplify a 1-kb product containing the aadA gene, which was cloned into the vector pCRII and transformed into Escherichia coli DH5-alpha competent cells. D-Rhodamine dye terminator cycle sequencing was used to determine the gene sequence, which was compared to previously reported sequences of aadA genes. We found the aadA gene in E. faecalis to be identical to the aadA genes reported by Sundstr om et al. for E. coli plasmid R6-5 (L. Sundström, P. Râdström, G. Swedberg, and O. Sköld, Mol. Gen. Genet. 213:191-201, 1988), by Fling et al. for the aadA within transposon Tn7 (M. E. Fling, J. Kopf, and C. Richards, Nucleic Acids Res. 13:7095-7106, 1985), and by Hollingshead and Vapnek for E. coli R538-1 (S. Hollingshead and D. Vapnek, Plasmid 13:17-30, 1985). Previous reports of the presence of the aadA gene in enterococci appear to be erroneous and probably describe an aadE gene, since the isolates were reported to be susceptible to spectinomycin.

[Infections caused by multiresistant enterococci in Norway]. / Infeksjoner forårsaket av multiresistente enterokokker i Norge.

During the last decade antimicrobial resistant pathogens have become a major medical problem. Internationally, multiresistant enterococci have increased nosocomial morbidity and mortality. Such strains are often resistant to ampicillin, aminoglycosides, and glycopeptides such as vancomycin. The spread of these strains has been shown to correlate to the use of antibiotics and the practice of suboptimal infection control within health care facilities. The current situation in Norwegian hospitals is presented, including the only six cases with infections and the three carriers of vancomycin resistant enterococci found to date. Surveillance in the hospitals shows that such strains are uncommon in non-infected patients. To maintain this favourable situation it is necessary to continue to practice effective methods of infection control and to employ sound antibiotic policies.

Antibodies to orthopoxvirus in domestic cats in Norway.

The prevalence of antibodies to orthopoxvirus in 217 sera collected from domestic cats in the western part of Norway was 10.1 per cent as measured by a competitive ELISA. In one of the seropositive cats antibodies were also detected by an immunofluorescence assay. The average age of the cats sampled was 4.9 years, but the average age of the seropositive individuals was 7.3 years, higher than the average age of clinical cowpox virus cases in Britain (4.2 years), and in Germany (3.9 years). Antibodies against feline immunodeficiency virus (FIV) were detected in nine of 30 (30 per cent) of the seropositive cats, and in five of 30 (17 per cent) of the seronegative cats, which suggests that FIV infection may influence the susceptibility of domestic cats to orthopoxvirus, or vice versa. Orthopoxvirus infections, have recently been detected in rodent populations in several areas of Norway, and the infection may therefore be present in cats all over the country; cat owners and animal handlers should be aware of this (re)emerging zoonosis.

Characteristics of four cowpox virus isolates from Norway and Sweden.

We report the first isolation of cowpox virus from a domestic cat in Norway, and the first confirmed isolation of cowpox virus from a human case in Norway. These two Norwegian cowpox virus isolates, as well as two Swedish human isolates, were partially characterized and compared with each other and with cowpox virus Brighton and vaccinia virus strain Western Reserve. Restriction enzyme analysis of the genomes revealed differences between all six viruses examined, but suggested that the two Norwegian isolates are closely related, as are the two Swedish isolates. Restriction endonuclease digestion of genomic DNA demonstrated that one of the Swedish isolates and the two Norwegian isolates have larger genomes than vaccinia virus strain Western Reserve, but smaller than cowpox Brighton. All four Scandinavian isolates lacked a 72 base-pair region within the A-type inclusion body protein gene which is present in the prototype cowpox virus Brighton.

Naturally occurring orthopoxviruses: potential for recombination with vaccine vectors.

Orthopoxviruses are being increasingly used as live recombinant vectors for vaccination against numerous infectious diseases in humans, domestic animals, and wildlife. For risk assessments and surveillance, information about the occurrence, distribution and ecology of orthopoxviruses in western Europe is important but has mainly been based on serological investigations. We have examined kidneys, lungs, spleens, and livers of Norwegian small rodents and common shrews (Sorex araneus) for the presence of orthopoxvirus DNA sequences by PCR with primers complementary to the viral thymidine kinase (TK) gene. PCR amplicons were verified as orthopoxvirus specific by hybridization with a vaccinia virus TK-specific probe. A total of 347 animals (1,388 organs) from eight locations in different parts of Norway, collected at different times of the year during 1993 to 1995, were examined. Fifty-two animals (15%) from five locations, up to 1,600 km apart, carried orthopoxvirus DNA in one or more of their organs, most frequently in the lungs. These included 9 of 68 (13%) bank voles (Clethrionomys glareolus), 4 of 13 (31%) gray-sided voles (Clethrionomys rufocanus), 3 of 11 (27%) northern red-backed voles (Clethrionomys rutilus), 16 of 76 (21%) wood mice (Apodemus sylvaticus), and 20 of 157 (13%) common shrews. The previous isolation of cowpox virus from two clinical cases of infection (human and feline) at two of the locations investigated suggests that the viruses detected are cowpox and that some of the virus-carrying small mammalian species should be included among the cowpox virus natural reservoir hosts in Scandinavia and western Europe.

Antibodies against orthopoxviruses in wild carnivores from Fennoscandia.

Two hundred and three sera obtained in 1993-96 from red foxes (Vulpes vulpes), lynx (Lynx lynx), brown bears (Ursus arctos) and wolverines (Gulo gulo) in Fennoscandia (Norway, Sweden, and Finland) were examined for the presence of anti-orthopoxvirus antibodies by a competition enzyme linked immunosorbent assay (ELISA). High prevalences were found for the red foxes in Norway (7/62, 11%) and Finland (7/14, 50%). While only one of 73 (1%) lynx from Finland had anti-orthopoxvirus antibodies, a high prevalence was found in sera from the Sarek National Park in Sweden (5/17, 29%). In addition, anti-orthopoxvirus antibodies were found in one brown bear from the same area (1/45, 2%), whereas none of the 14 wolverines were seropositive. This is the first report of anti-orthopoxvirus antibodies in the brown bear and the lynx, and the first screening for such antibodies in Sweden and Finland. These results indicate that orthopoxviruses are distributed in Sweden and Finland as well as in Norway, and that the red fox and the European lynx may serve as indicator species for the presence of orthopoxviruses in the local populations of small mammals.

[First infection with vancomycin resistant type VanA enterococci in a Norwegian hospital]. / Første infeksjon med vankomycinresistente enterokokker type VanA i et norsk sykehus.

Enterococci are part of the normal human and animal bowel flora. They are considered bacteria of relatively low virulence, but are important nosocomial pathogens. In the context of their intrinsic resistance to a number of antimicrobial agents, the rapid emergence of multiresistant enterococci is alarming. As inhabitants of the gastrointestinal tract, they come into close contact with other bacteria and may pass antibiotic resistance genes to them. We report the first case of infection with a VanA vancomycin-resistant. Enterococcus in Norway. The strain was identified as Enterococcus faccium with high level resistance to aminoglycosides, ampicillin, teicoplanin and vancomycin. The VanA phenotype was confirmed by PCR detection of the vanA gene. Transmission, treatment, prevention, and control of infections with vancomycin-resistant enterococci is discussed.

Serosurvey for orthopoxviruses in rodents and shrews from Norway.

Two hundred and twenty one blood samples representing eight different rodent species and the common shrew (Sorex araneus), collected in Norway between 1993 and 1995, were examined for anti-orthopoxvirus antibodies by a competition enzyme linked imunnosorbent assay (ELISA) and, when possible, an indirect immunofluorescence assay. The serological results indicated that the bank vole (Clethrionomys glareolus), woodmouse (Apodemus sylvaticus) and Norway lemming (Lemmus lemmus) may be reservoir species for orthopoxviruses in Norway, with antibody prevalences of 17 (12/69), 30 (24/81) and 56% (19/34), respectively. Orthopoxvirus infection in lemmings has not been reported previously. On some other small rodent species such as field voles (Microtus agrestis), common rats (Rattus norvegicus), and common shrews, seropositive individuals were detected. However, the total number of tested animals was low, and the role of these species in the epidemiology of orthopoxvirus infections remains unclear. Attempts to isolate orthopoxviruses from these small mammals failed, although orthopoxvirus specific DNA sequences were detected previously in the same animals by the polymerase chain reaction (PCR). The serological results were compared with and discussed in the context of the occurrence of orthopoxvirus-specific DNA sequences, and it is concluded that orthopoxviruses are widely distributed among wildlife in Norway.

Long PCRs of transposons in the structural analysis of genes encoding acquired glycopeptide resistance in enterococci.

Glycopeptide-resistant enterococci (GRE) associated with multiple antibiotic resistance present a major challenge to clinical practice and infection control due to limited or nonexistent antimicrobial treatment options. The genes encoding VanA- and VanB-type glycopeptide resistance have been shown to reside on transposons Tn1546 and Tn1547, respectively. These transferable genetic elements may carry the resistance determinants between and within different ecological niches. Molecular epidemiological studies of nosocomial outbreaks of VanA- and VanB-type GRE indicate horizontal transfer of glycopeptide resistance genes as an important mechanism for the spread of GRE. To target infection control and better understand the epidemiology of GRE, outbreak investigations and molecular epidemiological studies should therefore apply at least two different approaches, i.e., molecular-typing methods to analyze bacterial genomic heterogeneity and structural analysis of mobile resistance determinants. Here we describe the development and use of long PCRs in the structural analysis of vanA and vanB gene clusters in GRE.

Transmission of VanA-type vancomycin-resistant enterococci and vanA resistance elements between chicken and humans at avoparcin-exposed farms.

The genetical relatedness between epidemiologically linked fecal VRE strains from poultry farmers (n = 5) and their broilers (n = 7) at five avoparcin-exposed Norwegian farms was examined. Pulsed-field gel electrophoresis (PFGE) of bacterial chromosomal digests and structural analysis of vanA resistance elements was performed. Animal and human Enterococcus faecium strains at one farm were genetically closely related with indistinguishable vanA elements and a single band position difference in PFGE analysis. Examination of the vanA elements in genetically unrelated strains by restriction enzyme digestion of Tn1546 long-PCR amplicons and ORF2-vanR intergenic sequencing revealed a pool of at least two distinct vanA gene cluster groups in the two reservoirs. The results indicate that transmission of VanA glycopeptide resistance in enterococci between human and animal at avoparcin-exposed farms can occur by direct transfer of VRE strains as well as horizontal spread of resistance genes between strains.

Virulence properties of Escherichia coli isolated from Ethiopian patients with acute or persistent diarrhoea.

Escherichia coli strains isolated from faecal specimens of 108 Ethiopian patients with acute watery diarrhoea (n = 30), acute bloody (n = 9), and persistent (n = 25) diarrhoea, and from 44 patients who recently had recovered from diarrhoea were analyzed for the presence of virulence factors using DNA probes, and for adhesion to HeLa cells. Eighty-two patients were under five years of age. Enterotoxigenic E.coli (ETEC) were most frequently isolated (63 patients, 58%). Eighteen of the ETEC strains also hybridized with probes for EPEC adherence factor (EAF) and Enteroaggregative (EAgg) adherence. Enteroaggregative E. coli (EAggEC) were more frequently isolated than EAF positive E.coli, and more frequently from patients with persistent diarrhoea (10/25) than from patients with acute diarrhoea (11/39). In total, 103 of the patients harboured faecal E. coli which hybridized with one or more of the virulence probes. Haemagglutination of one or more erythrocyte species was expressed by 65/70 strains. Using monoclonal antibodies to Colonization Factor Antigen I and Coli Surface antigens 1-5, only 18/66 strains were found to produce one or more of these adhesions and no more than 15 of 43 ETEC strains were agglutinated by the antisera to these adhesins. Forty-nine strains adhered to HeLa cells in autoaggregative (23 strains), localized (17 strains) or diffuse (9 strains) pattern. The study shows that E.coli strains carrying genes for the different virulence factors are prevalent in Ethiopia. Testing for the presence of these virulence factors, as well as for putative colonization factor antigens, should be included in epidemiological studies in this area.

Low faecal carrier rate of vancomycin resistant enterococci in Norwegian hospital patients.

The faecal carrier rate of vancomycin resistant enterococci (VRE) was surveyed among 616 patients in selected departments of 7 Norwegian hospitals. One Enterococcus gallinarum isolate harbouring a vanB2 element was recovered from a child with malignant disease treated with vancomycin and ceftazidime. No vancomycin resistant Enterococcus faecalis or Enterococcus faecium were detected and no VRE isolates of the VanA type were identified. The low level of VRE carriage corresponds to the limited use of glycopeptide antibiotics for human therapeutic purposes in Norway. It indicates a low risk of acquiring VRE infections in Norwegian hospitals.

Evolutionary relationships among members of the genus Chlamydia based on 16S ribosomal DNA analysis.

Nucleotide sequences from strains of the four species currently in the genus Chlamydia, C. pecorum, C. pneumoniae, C. psittaci, and C. trachomatis were investigated. In vitro-amplified RNA genes of the ribosomal small subunit from 30 strains of C. pneumoniae and C. pecorum were subjected to solid-phase DNA sequencing of both strands. The human isolates of C. pneumoniae differed in only one position in the 16S rRNA gene, indicating genetic homogeneity among these strains. Interestingly, horse isolate N16 of C. pneumoniae was found to be closely related to the human isolates of this species, with a 98.9% nucleotide similarity between their 16S rRNA sequences. The type strain and koala isolates of C. pecorum were also found to be very similar to each other, possessing two different 16S rRNA sequences with only one-nucleotide difference. Furthermore, the C. pecorum strains truncated the 16S rRNA molecule by one nucleotide compared to the molecules of the other chlamydial species. This truncation was found to result in loss of a unilaterally bulged nucleotide, an attribute present in all other eubacteria. The phylogenetic structure of the genus Chlamydia was determined by analysis of 16S rRNA sequences. All phylogenetic trees revealed a distinct line of descent of the family Chlamydiaceae built of two main clusters which we denote the C. pneumoniae cluster and the C. psittaci cluster. The clusters were verified by bootstrap analysis of the trees and signature nucleotide analysis. The former cluster contained the human isolates of C. pneumoniae and equine strain N16. The latter cluster consisted of C. psittaci, C. pecorum, and C. trachomatis. The members of the C. pneumoniae cluster showed tight clustering and strain N16 is likely to be a subspecies of C. pneumoniae since these strains also share some antigenic cross-reactivity and clustering of major outer membrane protein gene sequences. C. psittaci and strain N16 branched early out of the respective cluster, and interestingly, their inclusion bodies do not stain with iodine. Furthermore, they also share less reliable features like normal elementary body morphology and plasmid content. Therefore, the branching order presented here is very likely a true reflection of evolution, with strain N16 of the species C. pneumoniae and C. psittaci forming early branches of their respective cluster and with C. trachomatis being the more recently evolved species within the genus Chlamydia.