Johan A. Maeland

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 staphyloccal methicillin resistance. A total of 219 staphylococcal strains were tested and the mPCR data were compared with coagulase production and in vitro oxacillin suscpetibility. The agreement was 100% for coagulase production and nuc amplification, and 97.7%, 96.8 and 97.3% for mecA amplication 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 μg/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.

Typing of human isolates of Streptococcus agalactiae (group B streptococcus, GBS) strains from Zimbabwe

Serotyping and genotyping are important tools in epidemiological studies of group B streptococcal (GBS) infections, which are important diseases in man, particularly in newborns. In the present study, 241 GBS isolates from Zimbabwe, comprising 124 carrier isolates from pregnant women and 117 isolates from patients hospitalised for various diseases, were serotyped. Antibodies specific for the capsular polysaccharide antigens (CPAs) Ia, Ib and II-V and antibodies specific for the surface-localised proteins, c(alpha), c(beta), R1, R3 and R4 were used for serotyping. Strains of the CPA types Ia (17%), III (47.7%) and V (23.2%) predominated. Of the various protein antigens, c(alpha) and R4 were expressed with highest frequency, c(alpha) by 100% of the CPA type Ia strains and R4 by 92% of the CPA type III strains. The R3 protein occurred frequently (24%), especially in type V strains (84%). A total of 25 serovariants was detected in the strain collection with the variants Ia/c(alpha) (16%), III/R4 (43.5%) and V/c(alpha), R3 (14.1%) occurring with the highest frequency. Serotype and subtype distribution of the carrier isolates were essentially similar to those of the disease-associated isolates. Genomic heterogeneity was demonstrated by pulsed-field gel electrophoresis of type III/R4 and type V/c(alpha), R3 isolates, but to a much lesser extent than recorded with Norwegian strains. These results demonstrate that many variants of GBS occur in the Zimbabwean population. The data obtained may assist in the formulation of a possible future GBS vaccine for Zimbabwe and perhaps for other African countries.

Mechanisms of methicillin resistance in staphylococci

The continuously high prevalence of methicillin‐resistant staphylococci (MRS) throughout the world is a constant threat to public health, owing to the multiresistant characteristics of these bacteria. Methicillin resistance is phenotypically associated with the presence of the penicillin‐binding protein 2a (PBP2a) not present in susceptible staphylococci. This protein has a low binding affinity for β‐lactam antibiotics. It is a transpeptidase which may take over cell wall synthesis during antibiotic treatment when normally occurring PBPs are inactivated by ligating β‐lactams. PBP2a is encoded by the mecA gene, which is located in mec, a foreign DNA region. Expression of PBP2a is regulated by proteins encoded by the plasmid‐borne blaR1‐blaI inducer‐repressor system and the corresponding genomic mecR1‐mecI system. The blaR1‐blaI products are important both for the regulation of β‐lactamase and for mecA expression. Methicillin resistance is influenced by a number of additional factors, e.g. the products of the chromosomal fem genes which are important in the synthesis of normal peptidoglycan precursor molecules. Inactivation of fem‐genes results in structurally deficient precursors which are not accepted as cell wall building blocks by the ligating PBP2a transpeptidase during antibiotic treatment. This may result in reduced resistance to β‐lactam antibiotics. Inactivation of genes affecting autolysis has shown that autolytic enzymes are also of importance in the expression of methicillin resistance. Methicillin resistance has evolved among earth microorganisms for protection against exogenous or endogenous antibiotics. Presumably the mec region was originally transferred from coagulase negative staphylococci (CNS) to Staphylococcus aureus (SA). A single or a few events of this kind with little subsequent interspecies transfer had been anticipated. However, recent data suggest a continuous horizontal acquisition by S. aureus of mec, being unidirectional from CNS to SA. Methicillin resistance may also be associated with mechanisms independent of mecA, resulting in borderline methicillin resistance. These mechanisms include β‐lactamase hyperproduction, production of methicillinases, acquisition of structurally modified normal PBPs, or the appearance of small colony variants of SA. Most MRS are multiresistant, and the mec region may harbour several resistance determinants, resulting in a clustering of resistance genes within this region.

Direct identification of Staphylococcus aureus in blood cultures by detection of the gene encoding the thermostable nuclease or the gene product

This study compares methods for direct identification of S. aureus in blood cultures by detection of the thermonuclease (TNase) of this bacterium or the nuc gene encoding it. The protein was detected by an enzyme diffusion test in o‐toluidine blue DNA agar with a test time of at least 4 h, by a monoclonal antibody (MAb)‐based sandwich enzyme‐linked immunosorbent assay (sELISA) with a test time of ˜4 h, and by a MAb‐based sandwich enzyme‐linked immunofiltration assay (sELIFA) with a test time of 25–30 min (sample preparation included). The nuc gene was amplified by a polymerase chain reaction (PCR) with a test time (amplification plus detection) of ‐3.5 h. The tests were optimized for direct examination of blood‐containing cultures. All tests were positive with 67/67 blood cultures which grew S. aureus, negative with 35/35 cultures which grew coagulase‐negative staphylococci, and negative with 37/37 cultures with various other bacteria. These results showed positive agreement with those of the commercial AccuProbe test but not with the StaphAurex agglutination kit. With an artificially seeded blood culture, minimum total times required (incubation plus testing) were as follows: nuc‐PCR, 9.5 h; sELIFA, 12.5 h; enzymatic test, 16–36 h; AccuProbe, 14 h. Direct examination of both the nuc gene and the mecA gene encoding methicillin resistance demonstrated the mecA gene in all the coagulase‐negative staphylococci (48.6%) which showed oxacillin resistance. The sELIFA had the particular advantage of its short test time, the PCR its high sensitivity and the possibility of simultaneous detection of the species‐specific nuc gene and genes encoding other clinically important characters of the bacteria. These tests offer the prospect of direct application to a variety of clinical specimens for rapid diagnosis of S. aureus infection.

Serum antibodies to cross‐reactive Neisseria outer membrane antigens in healthy persons and patients with meningococcal disease

Outer membranes (OMs) were prepared from the Neisseria meningitidis (Nm) strain 44/76, N. gonorrhoeae (Ng) NRL 8658, and N. lactamica (N1) ATCC 23970. Paired serum samples from 16 patients with serogroup B Nm disease and single samples from 30 blood donors were tested for IgG antibody levels against the three OMs in indirect ELISA, before and after absorption of the sera with N1 OM. Immunoblot analysis was used to identify OM target antigens for cross‐reacting and strain‐specific antibodies. Most of the Nm‐ and NG‐antibodies in sera from healthy adults were directed against OM antigens shared by the three Neisseria strains. Nm disease induced antibody formation against common Neisseria antigens, identified as the H.8 antigen and LPS determinants, against LPS determinants shared only by the Nm and Ng strains and against a variety of Nm‐specific OM antigens. Very low levels of Nm‐specific antibodies characterized the Nm patients in the acute phase. Also, the results indicate that the OM ELISA which has been used for the diagnosis of Nm disease, would be more useful if antibodies against common Neisseria antigens were removed from the sera before testing.

Streptococcus Agalactiae β Gene And Gene Product Variations

Streptococcus agalactiae (group B streptococci; GBS) are serotyped on the basis of the capsular polysaccharide antigens and subtyped on the basis of the strain-variable and surface-localised c proteins c α , c β , and R proteins. This study compared c β protein detection and the polymerase chain reaction (PCR) for β gene detection, by examining 50 clinical GBS strains. The c β protein was detected by antibody-based immunofluorescence in a GBS whole-cell assay and Western blotting by probing with the anti-c β antibody or human IgA. Absorption experiments were performed to test for surface-anchoring of c β ; and bacterial supernates were examined to test for c β production. Primers for the PCR target regions resulted in a 620-bp product that included β gene-encoding IgA-binding domains. The results demonstrated four categories of GBS with respect to the β gene and the c β protein: (1) strains (16 of 50) that harboured the β gene and regularly expressed normal surface-localised c β with a M r of 120 kDa; (2) strains (5 of 50) that harboured the gene but did not express the protein; (3) strains (2 of 50) that harboured the gene but expressed a c β that was not surface-localised and had reduced M r ; (4) strains (27 of 50) without β gene and c β expression. One strain amongst the third group generated a PCR product of 1330 bp. These results demonstrate considerable strain variability of the β gene of GBS and of its product the c β protein.

Susceptibility of skin and soft-tissue isolates of Staphylococcus aureus and Streptococcus pyogenes to topical antibiotics: indications of clonal spread of fusidic acid-resistant Staphylococcus aureus.

Staphylococcus aureus (SA) isolates (n=255) from outpatients with skin and soft-tissue infections were collected in 3 different areas in Norway. Group A streptococci (GAS, n=68) were isolated from skin or pharyngotonsillar specimens from outpatients. Minimum inhibitory concentrations (MIC) of bacitracin, fusidic acid and mupirocin were tested using the E-test. Pulsed field gel electrophoresis (PFGE) patterns of fusidic acid-sensitive (FusS) and -resistant (FusR) SA were compared. All GAS isolates showed MIC of bacitracin of =1.0 mg/l, of mupirocin of =0.125 mg/l and of fusidic acid 1.0–4.0 mg/l. All the SA showed MIC of mupirocin =0.5 mg/l and of bacitracin of =2.0 mg/l, 91% with MIC=16 mg/l. FusR was shown by 32.5% of the SA strains with similar prevalence rates in 3 different geographical areas of Norway. One particular PFGE pattern (type 1) was shown by 76% of the FusR SA. SA of type 1 belonged to phage group II and produced exfoliative toxins. Thus, the results demonstrated a high prevalence of FusR among SA causing skin infections and that this was mainly due to dissemination of clonally related FusR SA.

Distribution and expression of bca, the gene encoding the c alpha protein, by Streptococcus agalactiae.

A total of 52 clinical isolates of group B streptococci (GBS) was tested for expression of the c protein c(alpha) by a fluorescent antibody test (FAT) and by PCR amplification of a 202-bp stretch within the repeat unit of the bca gene encoding the c(alpha) protein. The strains were categorised as follows: c(alpha) FAT positive and PCR positive with amplification products of multiple sizes (category A, n = 12); FAT negative and with PCR products of multiple sizes (category B, n = 11); FAT negative and with a single PCR product of c. 200 bp (category C, n = 5); negative in both tests (category D, n = 24). A single amplification product of minimum size and additional products of larger sizes corresponded to one and more bca repeats, respectively. Five of the 11 category B strains showed expression of low Mr c(alpha) in whole cell-based Western blotting. The results showed that a proportion of the GBS isolates harboured bca gene elements that either were not expressed or they expressed c(alpha) molecular variants which could not be detected by the whole cell-based FAT. This genotype/phenotype discrepancy should be considered in relation to GBS typing, including the selection of antibody reagents and the technical approach to c(alpha) protein detection.

Distinctive Features of Surface-Anchored Proteins of Streptococcus agalactiae Strains from Zimbabwe Revealed by PCR and Dot Blotting

ABSTRACT The distribution of capsular polysaccharide (CPS) types and subtypes (serovariants) among 121 group B streptococcus (GBS) strains from Zimbabwe was examined. PCR was used for the detection of both CPS types and the surface-anchored and strain-variable proteins Cα, Cβ, Alp1, Alp2, Alp3, R4/Rib, and Alp4. The R3 protein was detected by an antibody-based method using monoclonal anti-R3 antibody in dot blotting. The CPS types detected, Ia (15.7% of strains), Ib (11.6%), II (8.3%), III (38.8%), V (24.0%), and nontypeable (1.7%), were essentially as expected on the basis of data from Western countries. The type V strains showed distinctive features with respect to protein markers in that Alp3 was detected in only 6.9% of the isolates while R3 occurred in 75.9% and R4/Rib occurred in 37.9% of the isolates. R3 occurred nearly always in combination with one of the alpha-like (Alp) proteins, and it was the third most common of the proteins studied. These results show that type V GBS strains from Zimbabwe differed from type V strains from other geographical areas and also emphasize the importance of the R3 protein in GBS serotyping and its potential importance in the immunobiology of GBS, including a potential role in a future GBS vaccine.

Properties and distribution of the putative R3 protein of Streptococcus agalactiae

Strain‐variable Streptococcus agalactiae (group B streptococci; GBS) proteins exposed at the bacterial cell surface are important markers in GBS serotyping. These proteins include the c proteins cα and cβ and the R proteins R1 through R4, of which R1 and R4 have been studied most extensively. This study presents the characteristics of a protein which was expressed by a capsular antigen type V GBS strain shown by means of polyclonal and monoclonal antibody testing. Examination of a number of reference and prototype strains by fluorescent antibody testing and Western blotting provided evidence that the serotype V‐derived protein was the R3 protein of GBS, previously defined on the basis of immunoprecipitation assays. The putative R3 protein formed ladder‐like banding patterns on Western blotting with polypeptides in the 30 kDa to ≥ 140 kDa range, was destroyed by pepsin digestion, and partially degraded by trypsin digestion. The protein was expressed by 10 (6.5%) of 153 clinical GBS strains tested, the expression being restricted to isolates of the capsular antigen types II, III, and V. Some isolates expressed both the cβ and the R3 protein. Expression in combination with cα or R4 protein synthesis was not detected. Inclusion of the anti‐R3 monoclonal antibody among antibody reagents for GBS serotyping will enhance the discriminatory power of this typing method.