Evaluating the usefulness of spa typing, in comparison with pulsed-field gel electrophoresis, for epidemiological typing of methicillin-resistant Staphylococcus aureus in a low-prevalence region in Sweden 2000-2004.

The usefulness of spa typing was evaluated in relation to pulsed-field gel electrophoresis (PFGE), as a tool for epidemiological typing of methicillin-resistant Staphylococcus aureus (MRSA) in a low-prevalence region in southern Sweden. Bacterial isolates from 216 MRSA cases, newly identified in 2000-2004, were studied. The isolates were obtained from infected patients (31%), and from colonized individuals found by screening (69%). In total, 49 spa types and 73 PFGE patterns were identified. The discriminatory power of spa typing was lower (94.9 +/- 1.8%) than that of PFGE (97.3 +/- 1.2%). For two spa types (t002 and t008) the Panton-Valentine leukocidin results added useful discriminatory information. The most common spa types were t044 (n = 31; four PFGE patterns), t002 (n = 24; 10 PFGE patterns), t067 (n = 12; four PFGE patterns), t050 (n = 12; one PFGE pattern), and t324 (n = 11; one PFGE pattern). Epidemiological investigations identified 91 single cases and 39 transmission chains, each involving two to 13 cases. All the transmission chains were held together both by spa and PFGE typing. Among the 91 single-case isolates, 33 spa types and 50 PFGE patterns were unique (matchless) at the time of identification. The low prevalence of MRSA, the low number of outbreaks, and the wide spectrum of strains due to frequent acquisitions abroad (49% of the cases), makes spa typing a useful complement to epidemiological investigations in our setting. However, we still recommend the continued use of PFGE for further discrimination of isolates with identical spa types when epidemiological data can not exclude possible transmission.

Validity of routine surveillance data: a case study on Swedish notifications of methicillin-resistant Staphylococcus aureus.

Surveillance of communicable diseases is a public health corner stone. Routine notification data on communicable diseases are used as a basis for public health action as well as for policy making. While there are agreed standards for evaluating the performance of surveillance systems, it is rarely possible to analyse the validity of the data entered into these systems. In this study we compared data on all Swedish cases of methicillin-resistant Staphylococcus aureus (MRSA) routinely notified between 2000 and 2003 with follow-up information collected for each of these cases as part of a public health project. The variables Reason for testing (clinical sample, contact tracing, screening of risk group), Clinical presentation (disease, colonisation), Transmission setting (healthcare-acquired, community-acquired), Country of acquisition (Sweden, abroad) and Risk-occupation (yes, no) were analysed for sensitivity, positive predictive value and completeness of answers. The sensitivity varied between 23% and 83%, the positive predictive values were generally higher (55% to 97%), while missing answers varied from 11% to 59%. The proportion of community-acquired cases was markedly higher when excluding either cases of MRSA colonisation or cases found through public health-initiated activities (contact tracing or screening of risk groups). We conclude that the quality of routine surveillance data may be inadequate for in-depth epidemiological analyses. This should be taken into account when interpreting routine surveillance figures. Whether or not the case definition includes cases of MRSA colonisation may have a significant impact on population-wide estimates of MRSA occurrence.

Guidelines for the validation and application of typing methods for use in bacterial epidemiology.

For bacterial typing to be useful, the development, validation and appropriate application of typing methods must follow unified criteria. Over a decade ago, ESGEM, the ESCMID (Europen Society for Clinical Microbiology and Infectious Diseases) Study Group on Epidemiological Markers, produced guidelines for optimal use and quality assessment of the then most frequently used typing procedures. We present here an update of these guidelines, taking into account the spectacular increase in the number and quality of typing methods made available over the past decade. Newer and older, phenotypic and genotypic methods for typing of all clinically relevant bacterial species are described according to their principles, advantages and disadvantages. Criteria for their evaluation and application and the interpretation of their results are proposed. Finally, the issues of reporting, standardisation, quality assessment and international networks are discussed. It must be emphasised that typing results can never stand alone and need to be interpreted in the context of all available epidemiological, clinical and demographical data relating to the infectious disease under investigation. A strategic effort on the part of all workers in the field is thus mandatory to combat emerging infectious diseases, as is financial support from national and international granting bodies and health authorities.

Staphylococcus aureus resistant to fusidic acid among Swedish children: a follow-up study.

We previously described the epidemic spread of a fusidic acid resistant clone of Staphylococcus aureus among children with bullous impetigo throughout Sweden 1. In this follow-up study we present data showing that Swedish clinicians in 2002 drastically reduced prescriptions of fusidic acid ointments, probably in response to the epidemic. Furthermore, we show that the fusidic acid resistant clone which dominated in 2001 is still the most prevalent fusidic acid resistant S. aureus in the population. The epidemic which reached its peak in 2002 is now clearly declining.

Report on the first PVL-positive community acquired MRSA strain in Latvia.

Infections by community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) have been reported worldwide. Here we present characterisation of the first CA-MRSA isolated in Latvia. A PVL-positive ST30-MRSA-IV strain was isolated from a nasal swab and the central venous catheter of a patient with fever and multiple organ failure. The PFGE pattern of this strain was identical to pattern SE00-3 of MRSA isolated in Sweden from 29 patients during 2000-2003. This strain is related to the South Pacific area, and its appearance in Sweden and Latvia demonstrates its global spread.

Diversity and evolution of the class A chromosomal beta-lactamase gene in Klebsiella pneumoniae.

We investigated the diversity of the chromosomal class A beta-lactamase gene in Klebsiella pneumoniae in order to study the evolution of the gene. A 789-bp portion was sequenced in a panel of 28 strains, representative of three phylogenetic groups, KpI, KpII, and KpIII, recently identified in K. pneumoniae and of different chromosomal beta-lactamase variants previously identified. Three groups of sequences were found, two of them corresponding to the families SHV (pI 7.6) and LEN (pI 7.1), respectively, and one, more heterogeneous, corresponding to a new family that we named OKP (for other K. pneumoniae beta-lactamase). Levels of susceptibility to ampicillin, cefuroxime, cefotaxime, ceftazidime, and aztreonam and inhibition by clavulanic acid were similar in the three groups. One new SHV variant, seven new LEN variants, and four OKP variants were identified. The OKP variants formed two subgroups based on nucleotide sequences, one with pIs of 7.8 and 8.1 and the other with pIs of 6.5 and 7.0. The nucleotide sequences of the housekeeping genes gyrA, coding for subunit A of gyrase, and mdh, coding for malate dehydrogenase, were also determined. Phylogenetic analysis of the three genes studied revealed parallel evolution, with the SHV, OKP, and LEN beta-lactamase families corresponding to the phylogenetic groups KpI, KpII, and KpIII, respectively. This correspondence was fully confirmed for 34 additional strains in PCR assays specific for the three beta-lactamase families. We estimated the time since divergence of the phylogenetic groups KpI and KpIII at between 6 and 28 million years, confirming the ancient presence of the beta-lactamase gene in the genome of K. pneumoniae.

Report on the first PVL-positive community acquired MRSA strain in Latvia.

Infections by community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) have been reported worldwide. Here we present characterisation of the first CA-MRSA isolated in Latvia. A PVL-positive ST30-MRSA-IV strain was isolated from a nasal swab and the central venous catheter of a patient with fever and multiple organ failure. The PFGE pattern of this strain was identical to pattern SE00-3 of MRSA isolated in Sweden from 29 patients during 2000-2003. This strain is related to the South Pacific area, and its appearance in Sweden and Latvia demonstrates its global spread.

Clonality among ampicillin-resistant Enterococcus faecium isolates in Sweden and relationship with ciprofloxacin resistance.

OBJECTIVE: To investigate clonal relationships in a nationwide sample of human Enterococcus faecium isolates. METHODS: Biochemical fingerprinting (PhP (PhenePlate) typing) was used to compare 180 fecal ampicillin-resistant E. faecium (ARE) isolates with 169 matched fecal ampicillin-susceptible E. faecium (ASE) isolates from patients in 23 hospitals, collected in 1998, and to study 39 fecal ARE isolates from non-hospitalized individuals collected in 1998, and five ARE and 29 ASE isolates from the early 1990s. Representative ARE and ASE isolates were subjected to pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA and sequencing of the regions encoding the fluoroquinolone targets of the enzymes GyrA and ParC. RESULTS: Both PhP and PFGE results showed a higher homogeneity among ARE than among ASE isolates (P < 0.001). One PhP type (FMSE1) comprised 73% of the hospital ARE isolates (53% of ARE isolates from non-hospitalized individuals, and four of five ARE isolates from the early 1990s), but only 1% of the ASE isolates. PFGE of the hospital E. faecium isolates revealed that 23 of the 25 ARE isolates and one of the 22 ASE isolates were of one dominating type. High-level resistance to ciprofloxacin (MIC > 16 mg/L) was present in 91% of ARE isolates, whereas only low-level resistance (MIC 4-16 mg/L; 35% of isolates) was found among ASE isolates. One mutation in parC (codon 80) and one of two mutations in gyrA (codons 83 or 87) were detected in all ARE isolates tested with high-level ciprofloxacin resistance, but were lacking in ARE and ASE isolates with low-level ciprofloxacin resistance. CONCLUSION: Most ARE isolates in Sweden were clonally related. High-level ciprofloxacin resistance was found in ARE isolates of PhP type FMSE1 as well as in other PhP types, but never in ASE isolates.

Clonal spread among Swedish children of a Staphylococcus aureus strain resistant to fusidic acid.

An increased incidence of fusidic acid-resistant Staphylococcus aureus strains causing superficial infections among children in Sweden has been noted since the mid-1990s. Based on routine susceptibility testing data from 10 laboratories representing 8/21 Swedish counties during 1990-2001, the increase was first demonstrated in southern Sweden and subsequently became apparent throughout the country. Epidemiological typing using pulsed-field gel electrophoresis of recent isolates of fusidic acid-resistant S. aureus from 11 laboratories representing 8/21 Swedish counties revealed a high degree of similarity of band patterns, indicating a clonal relationship. Data from 1 of the laboratories demonstrated a close connection between this clone and impetigo. Sales statistics showed a pronounced increase in the use of fusidic acid ointments in the 0-12 y age group from 1998 onwards. There was, however, no statistically significant correlation between sales of fusidic acid ointments and resistance among S. aureus strains to fusidic acid.

Control of an outbreak of a highly beta-lactam-resistant Enterobacter cloacae strain in a neonatal special care unit.

Two successive outbreaks of colonization and infection with Enterobacter cloacae resistant to third generation cephalosporins (cephalosporin-resistant E. cloacae, CREC) and involving 15 infants occurred within 12 months in a neonatal special care unit. Isolates of clinical significance were obtained from four infants (urine 2 cases, blood, pleural drainage). According to epidemiological typing using computerized biochemical fingerprinting and pulsed-field gel electrophoresis (PFGE) the same CREC strain was found in both outbreaks. The origin of the strain and its reservoir between the two outbreaks remained unknown. Emphasizing strict barrier nursing of the infants had little or no impact on the presence and transmission of the strain in the unit. In contrast, replacing ampicillin plus cefotaxime as standard empiric therapy with penicillin G plus netilmicin plus consequent cohorting of newborns and staff promptly halted both the outbreaks. During a 5-y follow-up after the last episode, the choice of antibiotics for empirical treatment has varied, and no further outbreaks of CREC have been seen, with the exception of two sporadic cases.

Transmission of urinary bacterial strains between patients with indwelling catheters--nursing in the same room and in separate rooms compared.

Despite lack of supporting scientific data it has been suggested that patients with an indwelling urinary catheter (IUC) should be nursed in separate rooms to reduce the risk of cross-infection. We conducted a one-month case-control study of nursing home patients with an IUC and bacteriuria, 20 nursed together pairwise and 20 in separate rooms, by weekly urine cultures and typing of the bacterial isolates. The transmission rate of urinary strains between patients was three times higher within rooms (5/9 possible transmissions) than between rooms (9/53 possible transmissions, P = 0.02). The study thus supported nursing IUC patients in separate rooms.

An allelic variant of the chromosomal gene for class A beta-lactamase K2, specific for Klebsiella pneumoniae, is the ancestor of SHV-1.

Fecal Klebsiella isolates from neonates in 22 Swedish special care units were examined by a PCR we developed for detection of the SHV-1 beta-lactamase gene. All 105 K. pneumoniae isolates and all 11 K. pneumoniae reference strains (including the K. pneumoniae subsp. pneumoniae, ozaenae, and rhinoscleromatis type strains) tested were positive, whereas all 67 K. oxytoca isolates and the K. oxytoca, K. planticola, and K. terrigena type strains tested were negative. Resistance to beta-lactams in K. pneumoniae was not transferable by conjugation, and the beta-lactamase gene was never found on a plasmid. Southern blot analysis showed that the gene had a defined chromosomal location. Isoelectric focusing and sequencing of 231-bp PCR amplicons from different isolates revealed many variants of the enzyme, with the two main groups being SHV-1 like (pI 7.6; 68 isolates) and LEN-1 like (pI 7.1; 14 isolates). Clavulanic acid markedly reduced the MICs of ampicillin for all the K. pneumoniae isolates tested. This fact, MIC profiles (penicillin rather than cephalosporin resistance), pIs, and sequence data showed that the chromosomal beta-lactamase of K. pneumoniae is a class A, group 2 enzyme distinct from the chromosomal AmpC enzymes found in several other gram-negative bacteria and from the chromosomal beta-lactamase K1 of K. oxytoca. We propose that the chromosomal beta-lactamase of K. pneumoniae be designated K2 and suggest that an allelic pI 7.6 variant of this enzyme is the ancestor of the SHV family of plasmid-mediated beta-lactamases.

A hospital outbreak of high-level beta-lactam-resistant Enterobacter spp.: association more with ampicillin and cephalosporin therapy than with nosocomial transmission.

We studied an 8 month outbreak of a 7-fold increased isolation rate of high-level beta-lactam-resistant Enterobacter spp. from clinical infections (20 patients, 22 isolates: 20 E. cloacae, 2 E. aerogenes). In a case-control analysis the occurrence of resistant Enterobacter spp. was found to be associated with treatment with multiple antibiotics (p = 0.03), broad-spectrum beta-lactam agents (p = 0.0001) including ampicillin (p = 0.04), and cephalosporins (cefuroxime and cefotaxime, p = 0.004). Biochemical fingerprinting and pulsed-field gel electrophoresis (PFGE) typing showed no identity between the resistant isolates, indicating that neither cross-infection nor nosocomial transmission from a common source was the immediate cause of the problem. The outbreak was not paralleled by the overall Enterobacter spp. isolation rate or the antibiotic usage pattern in the hospital. Thus, the underlying cause of the outbreak remained obscure.

Biochemical fingerprinting compared with ribotyping and pulsed-field gel electrophoresis of DNA for epidemiological typing of enterococci.

The Phene Plate (PhP) biochemical fingerprinting system for bacteria is based on measurements of the kinetics of bacterial biochemical reactions. This system was modified for typing of enterococci and was compared with DNA typing by pulsed-field gel electrophoresis and with ribotyping by using 45 Enterococcus faecalis isolates from international collections. It was also used to study 170 fecal enterococcal isolates from healthy individuals and 28 isolates of E. faecalis from the blood of neonates. The PhP system showed a high degree of discriminatory power for unrelated enterococcal isolates. Among the 170 unrelated fecal isolates, 107 isolates from international collections, PhP typing discriminated 19 types, and ribotyping discriminated 5 types. In most cases, when isolates were of the same DNA type, they were also of the same PhP type, and the level of agreement between these two methods was high (96%). A combination of PhP typing and DNA typing identified 34 different types, but ribotyping did not yield any further discrimination. PhP typing of E. faecalis isolates from healthy individuals (n = 89) and from the blood of neonates with septicemia (n = 28) yielded a diversity of 0.93 for both populations and similar major PhP types in both populations. Thus, the isolates from blood seemed to consist of a normal E. faecalis population, without a dominance of certain strains associated with virulence. We conclude that the PhP system is useful for epidemiological studies of enterococcal isolates, yielding results similar to those obtained with DNA typing by pulsed-field gel electrophoresis. Since PhP typing is a method that is simple and rapid and that is based on automatic evaluation of the data, it is suitable for analyzing large numbers of isolates and can be used alone or in combination with DNA typing or epidemiological and ecological studies of enterococci.

Epidemiology of plasmid-mediated beta-lactamases in enterobacteria Swedish neonatal wards and relation to antimicrobial therapy.

TEM-1, OXA-1, SHV-1, and related beta-lactamases in fecal isolates from 953 infants in 22 Swedish neonatal intensive care units were studied by DNA hybridization. TEM-1- and OXA-1-positive isolates were always Escherichia coli and represented 86 and 8%, respectively, of the ampicillin-resistant isolates of this species. SHV-1 was found in 16% of the Klebsiella sp. (mainly Klebsiella pneumoniae) isolates. TEM-1 and SHV-1 occurred in 14 and 16 units and in up to 64 and 26% of the neonates, respectively. On average, two to four different biochemical phenotypes per species per ward were positive for each beta-lactamase. All but 1 of the 33 E. coli phenotypes found to be TEM-1 positive were uniformly positive for the beta-lactamase gene, whereas some of the phenotypes found to be positive for OXA-1 (2 of 3) and SHV-1 (6 of 70) were occasionally negative for the respective genes. The occurrence of the three beta-lactamases studied tended to be associated with local ampicillin usage (correlation coefficient, 0.31 to 0.39; P greater than 0.05). Of the neonates receiving ampicillin, 30% carried TEM-1-positive E. coli, compared with 13% for cephalosporin-treated neonates and 15% for untreated neonates (P less than or equal to 0.001). The corresponding rates for SHV-1 in Klebsiella spp. were 18, 13, and 9% (P less than or equal to 0.01). Ampicillin is thus a significant risk factor for the maintenance of the most prevalent gram-negative plasmid-mediated beta-lactamases in hospitalized neonates.

Epidemiological typing of Corynebacterium diphtheriae isolated in Sweden 1984-1986.

From September 1984 to December 1986 121 cases of culture-positive diphtheria were reported to the National Bacteriological Laboratory in Stockholm. Toxigenic Corynebacterium diphtheriae nongravis was isolated from all but one of 33 patients with disease and from 69 healthy carriers. 63/65 toxigenic isolates, available for epidemiological typing, had the same phage type, 20, and the same restriction enzyme pattern, RE2. This included strains isolated both from patients inside and outside of the traditional risk groups of people abusing alcohol and drugs. Non-toxigenic strains gave different phage types and RE patterns.

The humoral antibody response to Shigella dysenteriae type 1 infection, as determined by ELISA.

An enzyme-linked immunosorbent assay (ELISA) for determining the class-specific humoral antibody response to the lipopolysaccharide antigen from Shigella dysenteriae serotype 1 bacteria has been tested. Two or more serum samples from each of 60 persons infected with this organism during a dysentery outbreak in a boarding school for young men near Haiphong, Viet Nam, and single serum samples from 39 healthy Vietnamese and from 20 healthy Swedes were included in the study. Comparison of the titres in the sera from the patients and the Vietnamese controls showed that the patients had significantly elevated IgA titres in sera collected 10, 30 and 45 days after onset of infection, and significantly elevated IgG titres in sera collected 30, 45 and 180 days after the onset. The titres in the patients' sera, compared with those in the Swedish controls, were significantly elevated for IgA and IgM as well as IgG in the samples collected after 10, 30, 45 and 180 days. The use of rabbit antisera, specific for enteropathogenic bacteria, and absorption experiments with human sera indicated that the S. dysenteriae type 1 lipopolysaccharide antigen is specific with respect to the O-antigenic polysaccharide chain.

Enzyme immunoassay of the antibody response to Brucella and Yersinia enterocolitica 09 infections in humans.

An enzyme immunoassay, with phenol-water extracted lipopolysaccharide (LPS) from Brucella abortus as antigen, was used to detect the class-specific antibody response in sera from 173 patients with B. abortus, B. melitensis or B. suis infection. Sera from 30 patients with salmonellosis, yersiniosis or tularaemia and from 25 healthy individuals served as controls. The B. abortus LPS antigen permitted a safe diagnosis of acute and chronic brucellosis with high IgM and rising IgG titres in sera collected in the acute stage of the disease, and with elevated IgG titres only in the chronic stage. The B. abortus LPS antigen also permitted a specific diagnosis with the exception of the high titres estimated in sera from patients with Yersinia enterocolitica 09 infection. The problem with that well-known reciprocal cross-reactivity was overcome by using two additional antigens: Y. enterocolitica 09 native and periodate oxidized and borohydride reduced LPS preparations. In sera from patients with brucellosis high titres were estimated against all three antigens, whereas in sera from patients with yersiniosis caused by serotype 09 high titres were measurable only with the B. abortus and the Y. enterocolitica native LPS antigens. These data suggest that the B. abortus and Y. enterocolitica 09 LPS share one antigenic determinant resistant to periodate oxidation and borohydride reduction, and that in addition the Y. enterocolitica 09 LPS has a determinant which is sensitive to periodate oxidation and borohydride reduction.