Multiplex polymerase chain reaction for detection of genes for Staphylococcus aureus thermonuclease and methicillin resistance and correlation with oxacillin resistance.

A multiplex polymerase chain reaction (mPCR) was used for simultaneous amplification of the staphylococcal nuc gene, encoding the thermostable nuclease (TNase), and the mecA gene, encoding the penicillin-binding protein 2a which is associated with staphylococcal methicillin resistance. A total of 219 staphylococcal strains were tested and the mPCR data were compared with coagulase production and in vitro oxacillin susceptibility. The agreement was 100% for coagulase production and nuc amplification, and 97.7%, 96.8 and 97.3% for mecA amplification and oxacillin resistance tested with MIC determination, disk diffusion and agar screen methods, respectively. Discrepant results were due to non-S. aureus isolates with borderline MICs of oxacillin (1-8 micrograms/ml). In a pilot test the mPCR simultaneously amplified both genes of staphylococci in blood cultures. This mPCR is a rapid and reliable method for single-step identification of cultures of MRSA and may prove to be useful for direct application on clinical specimens.

Comparison of various methods and reagents for species identification of Staphylococcus aureus positive or negative for the mecA gene.

The reliability of various methods for species identification of Staphylococcus aureus was evaluated. A total of 135 coagulase-positive (SA) or -negative (SS) staphylococcal isolates were tested, including methicillin-resistant (MR) and -susceptible (MS) strains. When the nuc gene which encodes the S. aureus thermonuclease (TNase) was amplified in a multiplex PCR simultaneously with the mecA gene which encodes for the MR-associated penicillin-binding protein 2a of staphylococci, the nuc amplification showed full agreement with the results of the coagulase test. TNase detected by an enzymatic method or as protein in a sandwich ELISA identified S. aureus with nearly the same precision as the PCR. The Staphylase, Monostaph and Staphaurex agglutination kits were all reliable for identification of MSSA, but not for MRSA. Most of the negative MRSA strains were identified by the Pastorex agglutination kit, in which reagents for fibrinogen receptor and protein A detection have been supplemented with antibodies for capsular polysaccharides of the serotypes 5 and 8. These results show that detection of the nuc gene or its TNase product is highly reliable for identification of both MRSA and MSSA strains, while various widely used agglutination kits do not show the same reliability for identification of MRSA strains.

Restriction endonuclease analysis of adenovirus type 3 isolated in Norway from 1970 to 1991.

Forty-three strains of adenovirus type 3 isolated from patients in Norway between 1970 and 1991 were analyzed with four restriction endonucleases. Bg1 II was the most discriminative enzyme. Five genotypes were identified and one of these has not been described before (Ad3a12). During both the epidemics in this period, new genotypes were introduced into the population. The same genotypes were identified in Norway as have previously been found in the northern parts of Europe, America and the Soviet Union.

Urinary tract infections in Norway: bacterial etiology and susceptibility. A retrospective study of clinical isolates.

OBJECTIVES: The objective of the present study was to determine bacterial etiology and susceptibility in urinary tract infections. The study was designed as a retrospective study of urine samples from patients both inside and outside hospitals and nursing homes that were received at our laboratory between 1 January 1997 and 31 October 1999. The Telemark Biomedical Center receives all the medical microbiology specimens from hospitals, nursing homes and general practitioners in the County of Telemark (165,000 inhabitants), Norway. All urines fulfilling the criteria for significant bacteriuria [> or =10,000 colony-forming units/mL urine] were included in the study. METHODS: Bacterial susceptibility testing was performed using breakpoint methodology. During the study period, we received 52 350 urine samples, of which 28,066 (53.6%) fulfilled the criteria for significant bacteriuria (pure growth of > or =10,000 CFU/mL urine). RESULTS: Escherichia coli was the most predominant bacterium in the urine from both inpatients (56.7%) and outpatients (68.3%). Coagulase-negative staphylococci and enterococci occurred significantly more often (P < 0.001) in urine samples from inpatients (12.5% and 7.9%) than in urine samples from outpatients (7.5% and 4.7%). Escherichia coli from both outpatients and hospitalised patients was highly susceptible (>93%) to cefalothin, mecillinam and nitrofurantoin, and more than 75% of E. coli isolates were also sensitive to ampicillin. Overall, the susceptibility to nitrofurantoin in bacteria from outpatients was 90% and from hospitalised patients was 85%. The corresponding figures for cefalothin were 92% and 90%, and for trimethoprim were 81% and 76%, respectively. CONCLUSIONS: Bacteria causing urinary tract infections in Norway are less resistant to antibacterial medication than in other western countries and the reason for this may be the low consumption of antibacterials by the Norwegian population. During the period from 1990 to 1999 the mean total annual consumption of antibacterial drugs in Norway was 15.3 defined daily doses per 1000 inhabitants per year.

Identification of Aerococcus urinae in urine samples.

To evaluate procedures for the identification of Aerococcus urinae, we examined 24 alpha-hemolytic non-enterococcal bacterial isolates from 4373 urine samples. Published procedures were compared with 16s rRNA sequencing and biochemical profiling (BBL-Crystal-GP). 16s rRNA sequencing and BBL-Crystal-GP identified the same 13 isolates as A. urinae. Published tests failed to distinguish the 13 A. urinae isolates from eight non-A. urinae isolates; several tests exhibited no discrimination. Ciprofloxacin and trimethoprim susceptibility and growth at 45 degrees C improved discrimination. For urinary isolates, standard procedures for identification of A. urinae are redundant and insufficiently discriminatory, and may need revision. BBL-Crystal-GP is an accurate alternative.

Heterogeneity of methicillin-resistant Staphylococcus aureus isolated in Norway.

Our objective was to look for differences in susceptibility patterns between Norwegian and imported methicillin-resistant Staphylococcus aureus (MRSA) strains. All MRSA isolates from the participating hospitals (87 isolates from 81 patients) throughout the period 1994-98 were examined, to study the clonal distribution of MRSA isolated in Norway and to identify any epidemic clones among the isolates. We found that imported isolates were resistant to an average of 5.6 antibiotics, while Norwegian isolates were resistant to an average of 2.6 antibiotics. MRSA isolates imported to Norway are more often multiresistant than domestic isolates. MRSA isolates in Norway show a striking diversity. Epidemic clones are present, but no single clone is predominant.

Comparison of PCR detection of mecA with agar dilution and Etest for oxacillin susceptibility testing in clinical isolates of coagulase-negative staphylococci.

Oxacillin-resistant staphylococci are heterogeneous in their expression of resistance to beta-lactam antibiotics. Different recommendations regarding screening methods for routine use have been published. In this study, the susceptibility to oxacillin of 232 coagulase-negative staphylococci (CoNS) was determined by agar dilution, Etest and presence of the mecA gene. When an oxacillin resistance breakpoint of > or = 0.5 mg/L was used, the sensitivity and specificity for agar dilution were 97.6% and 100%, and those for Etest were 100% and 95.4%. The current National Committee for Clinical Laboratory Standards oxacillin breakpoint recommendation will categorise accurately the CoNS species encountered commonly.

The prevalence of antibiotic resistance in bacterial respiratory pathogens from Norway is low.

OBJECTIVES: To determine the degree of bacterial susceptibility to the most commonly used drugs for respiratory infections in Norway, and to find if bacterial resistance is emerging. METHODS: Clinical isolates of Streptococcus pneumoniae, Haemophilus influenzae and group A streptococci from respiratory tract specimens and from the eye were collected from different parts of Norway during two study periods. During the first period (1993-1994), three laboratories, covering 15% of the Norwegian population, participated. During the second study period in 1997, five laboratories, covering 27% of the population, collected respiratory isolates. In total, 494 isolates of S. pneumoniae, 696 isolates of H. influenzae and 694 isolates of group A streptococci were included in the study. The study population comprised children and adults attending hospital and general practice. Bacterial susceptibility was determined by the E test, and breakpoints were according to the National Committee for Clinical Laboratory Standards (NCCLS). RESULTS: The prevalence of bacterial resistance was low, and we observed no significant increase in bacterial resistance between the two study periods. In 1997, only 0.6% of pneumococci had decreased susceptibility to penicillin, 1.6% of group A streptococci were resistant to erythromycin, and 6.7% of all isolates of H. influenzae produced beta-lactamase. CONCLUSIONS: The prevalence of antibiotic resistance in respiratory pathogens in Norway is low.

Laboratory handling of Helicobacter pylori critically influences the results of in-vitro metronidazole resistance determination.

In-vitro metronidazole resistance rates of Helicobacter pylori determined by Etest are high, and the predictive value of metronidazole resistance is low. It was hypothesised that altered laboratory methods could reduce the overestimation of resistance and improve the predictive value of the Etest. Pre-treatment isolates (n = 150) of H. pylori from 150 patients were investigated by Etest with incubation for 72 h. Treatment with metronidazole, tetracycline and bismuth for 10 days failed to eradicate H. pylori in 23 patients. After isolate storage for 3 years, resistance determination results by agar dilution and Etest, with incubation for 72 and 31 h, were compared. The rate of metronidazole resistance was reduced significantly during storage, and instability of resistance was associated significantly with treatment outcome. Isolates that retained in-vitro resistance had significantly (p 0.008) higher treatment failure rates (n = 13; 42%) than isolates that lost resistance (n = 3; 9%). The reproducibility achieved by dual testing with agar dilution and Etest was 41% and 70% for +/- 1 and +/- 2 log2 dilutions, respectively, after incubation for 72 h, and 85% and 92%, respectively, after incubation for 31 h. Thus, the predictive value was improved from 25% to 50% by the altered laboratory conditions (p 0.04). MIC values of 2-8 mg/L signified an intermediate risk of treatment failure.

Which contacts of patients with meningococcal disease carry the pathogenic strain of Neisseria meningitidis? A population based study.

OBJECTIVES: To determine the prevalence of the pathogenic strain of Neisseria meningitidis in contacts of patients with meningococcal disease, and to determine which contact groups are likely to be carriers and warrant chemoprophylaxis. DESIGN: Population based study. SETTING: Norwegian county of Telemark. SUBJECTS: 1535 primary contacts of 48 patients with meningococcal disease, and 78 secondary contacts. INTERVENTIONS: Carriers of the pathogenic strain were treated with rifampicin. All household members and kissing contacts under 15 years of age were treated with oral penicillin. Contacts were taught to recognise the symptoms of meningococcal disease. RESULTS: In 27 of 48 cases investigated, contacts carrying the pathogenic strain of N meningitidis were found. A total of 42 such contacts were identified. Contacts were stratified into three classes according to the assumed closeness of contact with patients. In class 1 (household members and kissing contacts) the prevalence of the pathogenic strain was 12.4% (95% confidence interval 5.5% to 19.3%). In classes 2 and 3 the prevalence was 1.9% (0.9% to 3.4%) and 1.6% (0.14% to 3.1%). CONCLUSIONS: There is a high rate of carriage of the pathogenic strain of N meningitidis in patients' household members and kissing contacts, and this supports the practice of giving chemoprophylaxis to these contacts. The prevalence of carriage among other contacts is 2-3 times that found in the general population (0.7%); the benefits of chemoprophylaxis to these contacts may be marginal.

Mutant ftsI genes in the emergence of penicillin-binding protein-mediated beta-lactam resistance in Haemophilus influenzae in Norway.

The most important mechanism for beta-lactam resistance in beta-lactamase-negative ampicillin-resistant (BLNAR) isolates of Haemophilus influenzae is the alteration of penicillin-binding protein 3 (PBP3) as a result of ftsI gene mutations. The present study aimed to map PBP3 alterations and to determine the correlation to beta-lactam resistance in respiratory tract isolates of H. influenzae in Norway, as well as assess the contribution of clonal spread to the emergence of PBP3-mediated resistance. Twenty-three beta-lactamase negative respiratory tract isolates with resistance to penicillins and 23 susceptible control isolates were examined by determination of beta-lactam MICs, ftsI sequencing and molecular typing by pulsed-field gel electrophoresis (PFGE). Ampicillin MIC ranges in the resistant group and the control group were 1-2 mg/L and 0.125-0.5 mg/L, respectively. All isolates in the resistant group had the PBP3 substitution Asn526-->Lys and were thus categorized as group II low-BLNAR. No control isolate met the genetic BLNAR (gBLNAR) criteria. The PBP3 substitution patterns corresponded well to those observed in previous European studies. Eighty-three percent (19/23) of the resistant isolates belonged to two clones, demonstrating the capability of low-BLNAR strains of clonal dissemination. Combined analysis of ftsI DNA sequences and PFGE patterns revealed distinctly different ftsI alleles in genetically indistinguishable isolates and identical copies of the same ftsI allele in unrelated isolates. A possible explanation of this observation is the recombinational exchange of ftsI alleles. This phenomenon, as well as the possibility of endemic European gBLNAR strains, should be further investigated.

Evaluation of new agglutination test for identification of oxacillin-susceptible and oxacillin-resistant Staphylococcus aureus.

A new agglutination test (Monostaph +; Bionor, Skien, Norway) has been developed. This new agglutination test has been compared with two other agglutination tests for the identification of 128 isolates of Staphylococcus aureus and 82 coagulase-negative staphylococci. The sensitivities of both Monostaph + and Pastorex Staph-Plus were excellent (98.7 and 97.4%, respectively) in detection of oxacillin-resistant Staphylococcus aureus. The specificity was 96.4% (two Staphylococcus epidermidis isolates and one Staphylococcus hominis isolate were false positive).

[Resistance development of major pathogenic bacteria]. / Resistensutvikling hos viktige sykdomsfremkallende bakterier.

In recent years the emergence of strains of drug-resistant bacteria has become a major health problem in many parts of the world. This has made it vitally necessary both to develop new antibacterial drugs and establish effective strategies to combat invading bacteria. In Norway, drug-resistant bacteria are a minor problem as yet, but the situation could change quite quickly if the necessary precautions are not taken. These include not prescribing antibacterial drugs too freely, and thoroughly surveying the extent of drug-resistance in an attempt to confine and prevent infection with resistant bacteria.

[Rat-bite fever]. / Rottebittfeber.

BACKGROUND: Rat bite fever and Haverhill fever are caused by Streptobacillus moniliformis which is part of the natural oral flora of rats and other rodents. Fever accompanied by headache, nausea and myalgia develops within ten days. Complications can be fatal. MATERIAL AND METHODS: A case of rat bite fever is described. A 48-year-old woman developed fever and malaise five days after being bitten by a pet rat. Two days later rash and arthritis in the hand and feet developed. Erythromycin was administered without effect. Ten days after the bite the patient was admitted to hospital and recovered after two weeks of intravenous penicillin therapy. RESULTS: S moniliformis was isolated from blood culture. On admission CRP was 231, ESR 88, ASAT 87, ALAT 218 and gamma-GT 461. Laboratory results normalized after therapy. INTERPRETATION: In cases of fever after rodent bites, S moniliformis infection should always be considered. Diagnosis is made by blood culture or cultivation from pus from the bite wound. First choice therapy is penicillin, or in case of intolerance, tetracycline.

[Infection outbreaks caused by methicillin-resistant Staphylococcus aureus at Haukeland hospital]. / Infeksjonsutbrudd med meticillinresistente gule stafylokokker ved Haukeland Sykehus.

BACKGROUND: Nosocomial infections caused by methicillin-resistant Staphylococcus aureus (MRSA) represent an increasing problem worldwide. MATERIAL AND METHODS: We report two outbreaks of methicillin-resistant S. aureus at Haukeland University Hospital during 1998-99. RESULTS: During the fall of 1998, four patients in the dermatological ward and three of their relatives were infected or colonised with MRSA. The strain was probably introduced by an eczematous patient who had recently arrived from Japan. Three patients became chronic carriers. The second outbreak involved three other hospital departments in July-August 1999. The index patient, a multitraumatised japanese tourist, died 16 days after admission. Two other patients were infected, one of them became a chronic carrier. According to official guidelines, neither of the index patients needed MRSA screening. Pulsed field gel-electrophoresis of the MRSA isolates revealed two different strains at the first outbreak, both probably introduced from the index patient, and one single strain at the second outbreak. Nasal swabs of staff were negative. INTERPRETATION: Physicians need to know that recommended guidelines regarding MRSA screening do not cover all types of risk situations. MRSA carriage among healthcare workers is probably not an important source of MRSA spread in hospitals. Measures to prevent cross infection between patients should be emphasised.

DNA fingerprinting of isolates of Staphylococcus aureus from newborns and their contacts.

During a study on the epidemiology of Staphylococcus aureus colonization in newborns, mothers, and hospital staff, S. aureus was isolated from 536 of 1,945 specimens. Ninety-three isolates of S. aureus from the three groups of individuals were included in a study to evaluate the potential of DNA fingerprinting for strain differentiation. The 93 isolates were also phage typed and their plasmid profiles were analyzed. Cleavage of DNA with BamHI resulted in 13 different DNA restriction endonuclease band patterns (DNA REBPs), one of which consisted of eight isolates whose DNA was not cleaved with BamHI. The DNAs from these eight isolates were easily cleaved with HindIII. The different DNA REBPs were stable both during in vitro and in vivo growth and allowed strain differentiation within phage groups or types. We could not show any strong association between DNA REBP classes, phage types or groups, and plasmid profiles. Of the 93 isolates, 27 (29.0%) could not be phage typed and 12 (12.9%) lacked plasmids. We therefore conclude that DNA fingerprinting is a powerful tool, in addition to phage typing and plasmid profile analysis, for strain differentiation of S. aureus.

[Human granulocytic ehrlichiosis in Norway]. / Human granulocytaer ehrlichiose i Norge.

BACKGROUND: The bacterium that causes human granulocytic ehrlichiosis may be transmitted by ticks. MATERIAL AND METHODS: We describe two patients with human granulocytic ehrlichiosis. During the summer of 1998, both patients were bitten by ticks. Four to 7 days later they developed influenza-like symptoms with fever, headache and myalgia. After 4 and 21 days, respectively, both patients were given doxycycline for suspected bacterial respiratory diseases, and recovered. RESULTS: Blood samples for human granulocytic ehrlichiosis antibodies showed a fourfold increase in titer in one patient and a remaining high titer in the other. Both patients had a positive polymerase chain reaction with primers specific for the Ehrlichia phagocytophilae genogroup. INTERPRETATION: The two patients fulfill the human granulocytic ehrlichiosis diagnostic criteria set by Centers for Disease Controls and Prevention, and are the first two human granulocytic ehrlichiosis cases described in Norway.

[Meningococcal project Telemark. Experiences after 5 years with contact tracing and eradication of the pathogenic bacteria in near contacts of the patients with meningococcal disease]. / Meningokokkprosjekt Telemark. Erfaringer etter fem år med smitteoppsporing og sanering av den sykdomsfremkallende bakterien hos naerkontakter av pasienter med meningokokksykdom.

Since 1987 we have analysed throat samples from 1,086 healthy contacts of 32 patients with meningococcal disease. The disease-causing strain was found in contacts of 17 out of the 32 patients. 161 (18%) of the contacts carried meningococci, and 30 (3%) of them were carriers of the disease-causing strain as determined by DNA fingerprinting. The carrier strain was eradicated in 29 of these 30 contacts by treatment with rifampicin. No secondary case of meningococcal disease was observed. During the four-year period 1984-87, there were 39 confirmed cases of meningococcal disease, including 12 verified and four suspected secondary cases of meningococcal disease. Therefore identification and eradication of the disease-causing strain seems to prevent secondary cases. A change in the Norwegian recommendations for preventing secondary cases of meningococcal disease should be discussed.

Preventing secondary cases of meningococcal disease by identifying and eradicating disease-causing strains in close contacts of patients.

In Norway, the use of chemoprophylaxis after cases of meningococcal disease is not recommended. Instead, household members less than 15 years are treated with penicillin for 7 days. Failures of this treatment have been reported. We therefore used DNA fingerprinting to identify the disease-causing strain in healthy contacts combined with selective rifampicin prophylaxis to these carriers to prevent secondary cases. During a 2-year period (1987-89) there were 13 cases of meningococcal disease in the County of Telemark (165000 inhabitants). 65 (14.7%) out of 441 contacts to these 13 patients harbored meningococci in their throat; 16 (3.6%) carried the disease-causing strain. Only 1 carrier fulfilled the criteria for being treated with penicillin; 8 were adults and the remaining 7 were not household members. No secondary cases of meningococcal disease occurred during the study period or the following 12 months. During the 4-year period (1984-87) preceding the study period there were 39 cases of meningococcal disease in Telemark; 7 of them were index cases for 12 bacteriologically verified and 4 clinically suspected secondary cases of meningococcal disease. We conclude that selective prophylaxis with rifampicin seems to be more efficient that penicillin treatment of household members less than 15 to prevent secondary cases of meningococcal disease.

[Examination of the environment in meningococcal disease. Identification and clearing of the virulent microorganism from the environment of patients with meningococcal disease]. / Miljøundersøkelse ved meningokokksykdom. Oppsporing og sanering av den sykdomsfremkallende mikrobe i miljøet til pasienter med meningokokksykdom.

During the period from January 1987 to June 1988 DNA fingerprinting was used to identify carriers of the disease-causing strain of Neisseria meningitidis among the patients contacts. A total of 432 persons were screened during nine episodes. The overall carrier-rate was 16.2%, and the causative strain was found in 3.4% of the contacts. Eleven carriers were successfully treated with rifampicin, whereas two of three carriers treated with penicillin remained carriers. We conclude that DNA fingerprinting is a valuable tool for rapid identification of carriers of the causative organism in order to eradicate the epidemic strain of N. meningitidis.