A. van Belkum

New Real-Time PCR Assay for Rapid Detection of Methicillin- Resistant Staphylococcus aureus Directly from Specimens Containing a Mixture of Staphylococci

ABSTRACT Molecular methods for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) are generally based on the detection of an S. aureus-specific gene target and the mecA gene. However, such methods cannot be applied for the direct detection of MRSA from nonsterile specimens such as nasal samples without the previous isolation, capture, or enrichment of MRSA because these samples often contain both coagulase-negative staphylococci (CoNS) and S. aureus, either of which can carry mecA. In this study, we describe a real-time multiplex PCR assay which allows the detection of MRSA directly from clinical specimens containing a mixture of staphylococci in <1 h. Five primers specific to the different staphylococcal cassette chromosome mec (SCCmec) right extremity sequences, including three new sequences, were used in combination with a primer and three molecular beacon probes specific to the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. Of the 1,657 MRSA isolates tested, 1,636 (98.7%) were detected with the PCR assay, whereas 26 of 569 (4.6%) methicillin-susceptible S. aureus (MSSA) strains were misidentified as MRSA. None of the 62 nonstaphylococcal bacterial species or the 212 methicillin-resistant or 74 methicillin-susceptible CoNS strains (MRCoNS and MSCoNS, respectively) were detected by the assay. The amplification of MRSA was not inhibited in the presence of high copy numbers of MSSA, MRCoNS, or MSCoNS. The analytical sensitivity of the PCR assay, as evaluated with MRSA-negative nasal specimens containing a mixture of MSSA, MRCoNS, and MSCoNS spiked with MRSA, was ∼25 CFU per nasal sample. This real-time PCR assay represents a rapid and powerful method which can be used for the detection of MRSA directly from specimens containing a mixture of staphylococci.

Colonisation by Streptococcus pneumoniae and Staphylococcus aureus in healthy children

A trial with a 7-valent pneumococcal-conjugate vaccine in children with recurrent acute otitis media showed a shift in pneumococcal colonisation towards non-vaccine serotypes and an increase in Staphylococcus aureus-related acute otitis media after vaccination. We investigated prevalence and determinants of nasopharyngeal carriage of Streptococcus pneumoniae and S aureus in 3198 healthy children aged 1-19 years. Nasopharyngeal carriage of S pneumoniae was detected in 598 (19%) children, and was affected by age (peak incidence at 3 years) and day-care attendance (odds ratio [OR] 2.14, 95% CI 1.44-3.18). S aureus carriage was affected by age (peak incidence at 10 years) and male sex (OR 1.46, 1.25-1.70). Serotyping showed 42% vaccine type pneumococci. We noted a negative correlation for co-colonisation of S aureus and vaccine-type pneumococci (OR 0.68, 0.48-0.94), but not for S aureus and non-vaccine serotypes. These findings suggest a natural competition between colonisation with vaccine-type pneumococci and S aureus, which might explain the increase in S aureus-related otitis media after vaccination.

Surgical Site Infections in Orthopedic Surgery: The Effect of Mupirocin Nasal Ointment in a Double-Blind, Randomized, Placebo-Controlled Study

The objective of this study was to determine whether use of mupirocin nasal ointment for perioperative eradication of Staphylococcus aureus nasal carriage is effective in preventing the development of surgical site infections (SSIs). A randomized, double-blind, placebo-controlled design was used. Either mupirocin or placebo nasal ointment was applied twice daily to 614 assessable patients from the day of admission to the hospital until the day of surgery. A total of 315 and 299 patients were randomized to receive mupirocin and placebo, respectively. Eradication of nasal carriage was significantly more effective in the mupirocin group (eradication rate, 83.5% versus 27.8%). In the mupirocin group, the rate of endogenous S. aureus infections was 5 times lower than in the placebo group (0.3% and 1.7%, respectively; relative risk, 0.19; 95% confidence interval, 0.02-1.62). Mupirocin nasal ointment did not reduce the SSI rate (by S. aureus) or the duration of hospital stay.

Nosocomial colonization and infection with multiresistant Acinetobacter baumannii: outbreak delineation using DNA macrorestriction analysis and PCR-fingerprinting

The prevalence of nosocomial acinetobacter colonization and infection in a university hospital was reviewed and multiresistant Acinetobacter baumannii infections in an intensive care unit (ICU) were investigated using epidemiological typing and a case-control study. Acinetobacter colonization at various body sites was found in 3.2 to 10.8 per 1000 patients. Acinetobacter infection accounted for 0.3% of endemic nosocomial infections in critically ill patients and for 1% of nosocomial bacteraemia hospitalwide. Over a three-week period, four ventilated patients developed colonization, followed by pneumonia in two patients, with A. baumannii resistant to multiple antimicrobials. Cultures of samples from respiratory equipment and ICU surfaces (n = 27) as well as from hands of personnel (n = 14) failed to yield A. baumannii, except for one sample of respiratory tubing. Antibiogram, biotype, chromosomal DNA macrorestriction profiles and polymerase chain reaction (PCR) mediated fingerprints of A. baumannii isolates (n = 31) indicated that this outbreak was caused by two strains, one of which later spread to another hospital where it caused a second outbreak. Both strains were clearly discriminated from control strains from cases of sporadic infection. Risk factors for cross-colonization that were identified by a case-control comparison were neurosurgery, mechanical ventilation and treatment with broad-spectrum antibiotics. Transmission was controlled by implementing contact isolation precautions and routine sterilization of ventilator tubing. Wider use of sensitive genotypic methods like DNA macrorestriction analysis and PCR-mediated fingerprinting for typing nosocomial pathogens should improve the detection of micro-epidemics amenable to early control.

Comparison of Staphylococcus aureus Isolates from Bovine and Human Skin, Milking Equipment, and Bovine Milk by Phage Typing, Pulsed-Field Gel Electrophoresis, and Binary Typing

ABSTRACT Staphylococcus aureus isolates (n = 225) from bovine teat skin, human skin, milking equipment, and bovine milk were fingerprinted by pulsed-field gel electrophoresis (PFGE). Strains were compared to assess the role of skin and milking equipment as sources of S. aureus mastitis. PFGE of SmaI-digested genomic DNA identified 24 main types and 17 subtypes among isolates from 43 herds and discriminated between isolates from bovine teat skin and milk. Earlier, phage typing (L. K. Fox, M. Gershmann, D. D. Hancock, and C. T. Hutton, Cornell Vet. 81:183-193, 1991) had failed to discriminate between isolates from skin and milk. Skin isolates from humans belonged to the same pulsotypes as skin isolates from cows. Milking equipment harbored strains from skin as well as strains from milk. We conclude that S. aureus strains from skin and from milk can both be transmitted via the milking machine, but that skin strains are not an important source of intramammary S. aureus infections in dairy cows. A subset of 142 isolates was characterized by binary typing with DNA probes developed for typing of human S. aureus. Typeability and overall concordance with epidemiological data were lower for binary typing than for PFGE while discriminatory powers were similar. Within several PFGE types, binary typing discriminated between main types and subtypes and between isolates from different herds or sources. Thus, binary typing is not suitable as replacement for PFGE but may be useful in combination with PFGE to refine strain differentiation.

Intestinal carriage of Staphylococcus aureus: how does its frequency compare with that of nasal carriage and what is its clinical impact?

The bacterial species Staphylococcus aureus, including its methicillin-resistant variant (MRSA), finds its primary ecological niche in the human nose, but is also able to colonize the intestines and the perineal region. Intestinal carriage has not been widely investigated despite its potential clinical impact. This review summarizes literature on the topic and sketches the current state of affairs from a microbiological and infectious diseases’ perspective. Major findings are that the average reported detection rate of intestinal carriage in healthy individuals and patients is 20% for S. aureus and 9% for MRSA, which is approximately half of that for nasal carriage. Nasal carriage seems to predispose to intestinal carriage, but sole intestinal carriage occurs relatively frequently and is observed in 1 out of 3 intestinal carriers, which provides a rationale to include intestinal screening for surveillance or in outbreak settings. Colonization of the intestinal tract with S. aureus at a young age occurs at a high frequency and may affect the host’s immune system. The frequency of intestinal carriage is generally underestimated and may significantly contribute to bacterial dissemination and subsequent risk of infections. Whether intestinal rather than nasal S. aureus carriage is a primary predictor for infections is still ill-defined.

Molecular typing of Cryptococcus neoformans : Taxonomic and epidemiological aspects

Pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) analysis, serotype, and killer toxin sensitivity patterns of a wide range of saprobic, clinical, and veterinary isolates of both varieties of Cryptococcus neoformans were examined. C. neoformans var. neoformans and C. neoformans var. gattii differed in chromosomal makeup, RAPD patterns, and killer sensitivity patterns. These results suggest that there are two separate species rather than two varieties. No clear genetic or phenotypic differences were observed among the clinical, saprobic, and veterinary isolates within each taxon. The serotypes differed substantially in their RAPD characteristics. Geographical clustering was observed among the isolates of C. neoformans var. gattii, but not among the isolates of C. neoformans var. neoformans. The isolates of each taxon that originated from restricted geographical areas often had identical or similar karyotypes and RAPD patterns, suggesting that clonal reproduction had occurred. The combination of PFGE and RAPD analysis allowed us to distinguish almost all isolates. This combination of techniques is recommended for further research on epidemiological, ecological, and population issues.

Short sequence repeats in microbial pathogenesis and evolution.

Abstract. Repetitive DNA is ubiquitous in microbial genomes. Different classes of short sequence repeats (SSRs) have been identified and demonstrated to be generally heterogeneous in a locus-dependent manner, reflected in variation in the number of repeat units present at a given genomic site or by sequence heterogeneity among individual units. Both types of variability can be used to assess intra-species genetic diversity. Repeat variability often affects the coding potential of the region in which the repetitive element is located. This implies that determination of the primary structure of variable numbers of tandem repeats can be used for epidemiological identification purposes, and also for the analysis of gene function. Precise assessment of SSR structure can also generate insight into the regulation of gene expression. Together, DNA repeat analysis in microbial species provides information on both functional and evolutionary aspects of genetic diversity among microbial isolates.Repetitive DNA is ubiquitous in microbial genomes. Different classes of short sequence repeats (SSRs) have been identified and demonstrated to be generally heterogeneous in a locus-dependent manner, reflected in variation in the number of repeat units present at a given genomic site or by sequence heterogeneity among individual units. Both types of variability can be used to assess intra-species genetic diversity. Repeat variability often affects the coding potential of the region in which the repetitive element is located. This implies that determination of the primary structure of variable numbers of tandem repeats can be used for epidemiological identification purposes, and also for the analysis of gene function. Precise assessment of SSR structure can also generate insight into the regulation of gene expression. Together, DNA repeat analysis in microbial species provides information on both functional and evolutionary aspects of genetic diversity among microbial isolates.

A novel link between Campylobacter jejuni bacteriophage defence, virulence and Guillain-Barré syndrome

Guillain–Barré syndrome (GBS) is a post-infectious disease in which the human peripheral nervous system is affected after infection by specific pathogenic bacteria, including Campylobacter jejuni. GBS is suggested to be provoked by molecular mimicry between sialylated lipooligosaccharide (LOS) structures on the cell envelope of these bacteria and ganglioside epitopes on the human peripheral nerves, resulting in autoimmune-driven nerve destruction. Earlier, the C. jejuni sialyltransferase (Cst-II) was found to be linked to GBS and demonstrated to be involved in the biosynthesis of the ganglioside-like LOS structures. Apart from a role in pathogenicity, we report here that Cst-II-generated ganglioside-like LOS structures confer efficient bacteriophage resistance in C. jejuni. By bioinformatic analysis, it is revealed that the presence of sialyltransferases in C. jejuni and other potential GBS-related pathogens correlated significantly with the apparent degeneration of an alternative anti-virus system: type II Clusters of Regularly Interspaced Short Palindromic Repeat and associated genes (CRISPR-Cas). Molecular analysis of the C. jejuni CRISPR-Cas system confirmed the bioinformatic investigation. CRISPR degeneration and mutations in the cas genes cas2, cas1 and csn1 were found to correlate with Cst-II sialyltransferase presence (p < 0.0001). Remarkably, type II CRISPR-Cas systems are mainly found in mammalian pathogens. To study the potential involvement of this system in pathogenicity, we inactivated the type II CRISPR-Cas marker gene csn1, which effectively reduced virulence in primarily cst-II-positive C. jejuni isolates. Our findings indicate a novel link between viral defence, virulence and GBS in a pathogenic bacterium.

Rapid Identification of Mycobacteria by Raman Spectroscopy

ABSTRACT A number of rapid identification methods have been developed to improve the accuracy for diagnosis of tuberculosis and to speed up the presumptive identification of Mycobacterium species. Most of these methods have been validated for a limited group of microorganisms only. Here, Raman spectroscopy was compared to 16S rRNA sequencing for the identification of Mycobacterium tuberculosis complex strains and the most frequently found strains of nontuberculous mycobacteria (NTM). A total of 63 strains, belonging to eight distinct species, were analyzed. The sensitivity of Raman spectroscopy for the identification of Mycobacterium species was 95.2%. All M. tuberculosis strains were correctly identified (7 of 7; 100%), as were 54 of 57 NTM strains (94%). The differentiation between M. tuberculosis and NTM was invariably correct for all strains. Moreover, the reproducibility of Raman spectroscopy was evaluated for killed mycobacteria (by heat and formalin) versus viable mycobacteria. The spectra of the heat-inactivated bacteria showed minimal differences compared to the spectra of viable mycobacteria. Therefore, the identification of mycobacteria appears possible without biosafety level 3 precautions. Raman spectroscopy provides a novel answer to the need for rapid species identification of cultured mycobacteria in a clinical diagnostic setting.