Identification of new phages to type Staphylococcus aureus strains and comparison with a genotypic method.

The aim of this study was to optimize the epidemiological monitoring of strains of Staphylococcus aureus, a major cause of hospital-acquired infections. From September to December 1998 47 S. aureus strains isolated from swabs taken from orthopaedic and trauma patients were in studied. Thirty-five isolates were sensitive to methicillin (MSSA) and 12 were methicillin-resistant (MRSA). Ten of the 47 isolates could not be phage-typed using the international set of typing phages: five of these isolates were MSSA and five were MRSA. These MRSA isolates, which were also not typeable by the phages currently recommended for phage-typing MRSA, were lysed by locally isolated experimental phages 584 and 1814. Phage 1814 lysed the gentamicin-resistant MRSA and phage 584 acted on the gentamicin-sensitive MRSA. Both new phages were inactive against the methicillin-sensitive isolates. Cloning of certain isolates was confirmed by macrorestriction genomic profiles obtained by pulsed-field gel electrophoresis analysis (PFGE). The results showed good discriminatory ability of antibiotic-resistance pattern phenotyping and phage-typing when the phages used were adapted to epidemic-associated MRSA strains.

Combined ribotyping and random multiprimer DNA analysis to probe the population structure of Listeria monocytogenes.

To improve our understanding of the genetic links between strains originating from food and strains responsible for human diseases, we studied the genetic diversity and population structure of 130 epidemiologically unrelated Listeria monocytogenes strains. Strains were isolated from different sources and ecosystems in which the bacterium is commonly found. We used rRNA gene restriction fragment length polymorphism analysis with two endonucleases and random multiprimer DNA analysis with seven oligonucleotide primers to study multiple genetic features of each strain. We used three clustering methods to identify genetic links between individual strains and to determine the precise genetic structure of the population. The combined results confirmed that L. monocytogenes strains can be divided into two major phylogenetic divisions. The method used allowed us to demonstrate that the genetic structure and diversity of the two phylogenetic divisions differ. Division I is the most homogeneous and can easily be divided into subgroups with dissimilarity distances of less than 0.30. Each of these subgroups mainly, or exclusively, contains a single serotype (1/2b, 4b, 3b, or 4a). The serotype 4a lineage appears to form a branch that is highly divergent from the phylogenetic group containing serotypes 1/2b, 4b, and 3b. Division II contains strains of serotypes 1/2a, 1/2c, and 3a. It exhibits more genetic diversity with no peculiar clustering. The fact that division II is more heterogeneous than division I suggests that division II evolved from a common ancestor earlier than division I. A significant association was found between division I and human strains, suggesting that strains from division I are better adapted to human hosts.

Multivariate analysis of determinants of bacterial contamination of whole-blood donations.

BACKGROUND AND OBJECTIVES: Introduction of bacteria into blood components at the collection stage seems to be a frequent occurrence. We therefore assessed determinants of bacterial contamination of whole-blood donations to gain insight into contamination mechanisms and direct prevention. MATERIALS AND METHODS: A cross-sectional study was carried out on donors accepted for whole-blood donation in four French blood banks. Each blood bank used its own two-stage procedure for phlebotomy site preparation. Contamination was identified by culturing two 15-ml samples (collected aseptically at the outset of donation) in a BacT/Alert 240 system. Determinants were assessed by logistic regression analysis. RESULTS: Bacterial contamination, mainly by skin flora, occurred in 76 (2.2%) out of 3385 donations. Significant determinants were as follows: the blood bank (odds ratio [OR] range = 3.0-5.6, P < 0.001); lack of repetition of scrub (OR = 2.7, P = 0.032); and donor age > 35 years (OR = 1.8, P = 0.036). CONCLUSION: Systematic scrub repetition should be implemented to reduce bacterial contamination by skin flora at the collection stage. Further research is required to clarify the role of different antiseptic agents and of donor age.

Efficacy of a new collection procedure for preventing bacterial contamination of whole-blood donations.

BACKGROUND: Transfusion-related bacterial contamination is a serious problem. The introduction of bacteria into donations at the collection stage seems frequent, despite well-conducted phlebotomy site preparation. Additional preventive measures are required. STUDY DESIGN AND METHODS: The aim of this study was to assess the potential efficacy of excluding the first 15 mL of blood to reduce the bacterial contamination of donations. A special device allowed the aseptic collection of two samples at the beginning of donation: S1 (first 15 mL) and S2 (next 15 mL). Bacteriologic cultures of S1 and S2 were performed by using an automated system. The procedure's efficacy was measured by the proportion of positive donations in S1 that were then negative in S2. RESULTS: S1 and/or S2 were positive in 76 (2.2%) of 3385 donations. In about three-fourths of the culture-positive donations, contamination was detected in the first 15-mL sample only. Gram-positive cocci accounted for 81 percent of species, gram-positive bacilli for 14 percent, and gram-negative bacilli for 5 percent. The new procedure would have prevented the introduction of bacteria in 55 donations, reducing to 0.6 percent the risk of contamination from the first 15 mL collected. CONCLUSION: Although the final effect on blood component bacterial contamination rates cannot be derived from the study, excluding the first 15 mL of blood may reduce the rate of bacterial contamination in donations.

Low sensitivity of Listeria monocytogenes to quaternary ammonium compounds.

Ninety-seven epidemiologically unrelated strains of Listeria monocytogenes were investigated for their sensitivities to quaternary ammonium compounds (benzalkonium chloride and cetrimide). The MICs for seven serogroup 1/2 strains were high. Three came from the environment and four came from food; none were isolated from human or animal samples. All 97 strains carried the mdrL gene, which encodes a multidrug efflux pump, and the orfA gene, a putative transcriptional repressor of mdrL. The absence of plasmids in four of the seven resistant strains and the conservation of resistance after plasmid curing suggested that the resistance genes are not plasmid borne. Moreover, PCR amplification and Southern blot hybridization experiments failed to find genes phylogenetically related to the qacA and smr genes, encoding multidrug efflux systems previously described for the genus Staphylococcus. The high association between nontypeability by phages and the loss of sensitivity to quaternary ammonium compounds are suggestive of an intrinsic resistance due to modifications in the cell wall.

Pseudomonas aeruginosa from cystic fibrosis patients: study using whole cell RAPD and antibiotic susceptibility.

Pseudomonas aeruginosa is the most important bacterial pathogen in lung disease of cystic fibrosis patients. Different morphotypes of the bacterium are frequently recovered in sputum samples of these patients. We developed a whole cell Randomly Amplified Polymorphic DNA (RAPD) technique in order to establish the relatedness between morphotype, genotype and antibiotic susceptibility. Six cystic fibrosis patients already colonized by P. aeruginosa were investigated by collecting three successive sputum samples (before and after antibiotic treatment, and one month later) and selecting 10 isolates per morphotype. 250 isolates of P. aeruginosa were recovered from 16 of 18 sputum samples. Five patients carried a single RAPD type strain four of which showed at least two morphotypes; one patient carried two RAPD types strains. No patients carried the same strain. These results confirmed other studies previously published in showing stability of the chronic colonization with a single strain. Antibiotype differences were not associated with differences of RAPD profiles and no relation was found between antibiotype and morphotype.

Evaluation of seven experimental phages for inclusion in the international phage set for the epidemiological typing of Listeria monocytogenes.

The purpose of our study was to evaluate the inclusion of seven experimental phages into the international phage set for subtyping Listeria monocytogenes. The seven additional phages included the broad-host-range virulent Myoviridae phage A511 (M. J. Loessner, Appl. Environ. Microbiol. 57:1912-1918, 1991), three temperate phages from the Danish subsystem for typing serotype 1/2 strains (12682, 6223, and 5775) (P. Gerner-Smidt, V.T. Rosdahl, and W. Frederiksen, APMIS 101:160-167, 1993), and three temperate phages isolated by this laboratory in France (9425, 1313, and 197). A panel of 395 Listeria monocytogenes isolates (including 180 that were non-phage typeable by the international set) were used in the study for a comparison of the lytic spectra of the various bacteriophages. These results showed that the inclusion of five of the experimental phages contributed greatly to the overall typeability and discriminatory power of the system, especially for strains within serogroup 1/2.

WHO study on subtyping Listeria monocytogenes: results of phage-typing.

A multi-centered study on phage typing of Listeria monocytogenes was carried out using 80 cultures sent under code and tested in six different laboratories. Phage typing was performed using an international phage set in five laboratories and phage sets unique to two laboratories. Testing of cultures sent in duplicate showed similar levels of reproducibility to that previously reported. Analysis of results from groups of epidemiologically related cultures showed a high level of agreement in all laboratories. Patterns of phage susceptibility were relatively stable on retesting strains in the same laboratory after long periods of time. However, there was limited comparability between results obtained from testing the same cultures using the same phages in different laboratories. It is recommended that the phages in the international set be reviewed, and that better inter-laboratory reproducibility may be achieved by standardisation of phage suspensions, propagation strains and methodology, together with the use of centrally propagated phages.

The WHO multicenter study on Listeria monocytogenes subtyping: random amplification of polymorphic DNA (RAPD).

As part of a WHO multicenter study on Listeria monocytogenes subtyping methods the random amplification of polymorphic DNA (RAPD)-technique was evaluated. Six participants were asked to use a standard protocol to analyse a set of 80 L. monocytogenes strains. This set contained 22 groups of epidemiologically linked isolates and 11 pairs of duplicate strains. Using three different 10-mer primers the median reproducibility of the RAPD-results obtained by the six participants was 86.5% (range 0-100%). Failure in reproducibility was mainly due to results obtained with one particular primer. The number of epidemiological groups found to be homogeneous varied from 1-22 (median 16). However, for some groups an inhomogeneity was found by the majority of participants. The overall correlation between the results from the different participants ranged from 32 to 85%.

[Informative capacity of 8 serological tests in the diagnosis of human brucellosis]. / Capacité informative de huit tests sérologiques dans le diagnostic de la brucellose humaine.

Assessment of the informative value of 8 immunological tests: sero-agglutination (Wright and Rose Bengale), indirect immunosorbent assay, counter immuno electrophoresis, ELISA IgG, IgM and IgA, and particle counting immunoassay (PACIA) has been performed among the results of serum of 209 patients. The patients were divided in four groups: 71 who already had brucellosis, 18 Yersinia infections, 12 Tularemia and 108 free of desease. The informative capacities of a positive result of counter immuno electrophoresis (Protein antigen Brucellin-INRA Tours-Nouzilly) is higher than others reactions and can be proposed as a confirmatory test of brucellosis. Among others techniques, 4 were found to be more sensitive: Elisa IgA (se = 97.6) and IgG (se = 90.1), IFI (se = 91.5) and Rose Bengale (se = 85.9) and can be proposed as screening test for medical diagnosis or epidemiological survey. Many cross-reactions were observed specially with Yersinia enterocolitica even with new serological methods.

Typing of Staphylococcus epidermidis strains by random amplification of polymorphic DNA.

The polymerase chain reaction was used to obtain randomly amplified polymorphic DNA profiles for typing of Staphylococcus epidermidis strains. Epidemiologically unrelated S. epidermidis isolates were screened with randomly amplified polymorphic DNA analysis. The discriminating ability of 45 randomly designed 10-mer primers was assessed. The highest discriminatory power was obtained with the 10-mer oligonucleotide OPAM-12. In typing a total of 13 unrelated S. epidermidis strains with OPAM-12, 11 different banding profiles were obtained reproducibly by agarose gel electrophoresis. The discriminatory power of the method with OPAM-12 was estimated using the D value of Hunter and Gaston (1988) to be 0.961. A reproducibility index of 1 was obtained after typing a total of 40 cultures including 12 triplicates and one quadruplicate of the 13 unrelated strains. Following the described procedure, the randomly amplified polymorphic DNA method provided a rapid, simple and reproducible alternative to other S. epidermidis typing systems.

Proposals for optimization of the international phage typing system for Listeria monocytogenes: combined analysis of phage lytic spectrum and variability of typing results.

Combined analysis of 5,179 serial phage reactions of 20 Listeria monocytogenes propagating strains over 14 years and phage typing results from 2,659 further L. monocytogenes strains allowed us to estimate lytic spectrum specificity and the variability of the lytic reactions of 35 phages. These included the 26 phages recommended for the international method for phage typing defined in 1985 by Rocourt et al. (J. Rocourt, A. Audurier, A. L. Courtieu, J. Durst, S. Ortel, A. Schrettenbrunner, and A. G. Taylor, Zentralbl. Bakteriol. Abt. 1 Orig. A 259:489-497, 1985). The results are discussed individually for each phage. Proposals for modifying the present system are made with the aim of producing an optimal bacteriophage set for routine use.

Epidemiological analysis of Acinetobacter baumannii strains isolated from a surgical intensive care unit.

A serious epidemic of Acinetobacter baumannii resistant to imipenem occurred in the surgical intensive care unit of the hospital Charles-Nicolle in Tunis during February 1994, causing two deaths among three patients. The Acinetobacter strains were isolated from various samples of the intensive care unit. The techniques used for typing were biotyping, antibiogram, plasmid profiles and chromosomal DNA by random amplified polymorphic DNA (RAPD). The A baumannii strains isolated from patients exhibited an identical pattern with all the epidemiological markers utilized; the strains from the surrounding areas showed four and six different patterns respectively for phenotypic and genotypic characters. The strain isolated from a care table had the same phenotypic and genotypic pattern as that of the patients' strains.

Changes in microbial ecology and use of cloxacillin.

There is generally a positive correlation between antibiotic consumption and incidence of resistance to antibiotics used either for prophylaxis or therapy in human infections. This was not the case for two surgical wards in our hospital. A 15-year study showed that the incidence of methicillin-resistant Staphylococcus aureus (MRSA) was unrelated to cloxacillin consumption, and in fact fell after introduction into the two wards of an antibiotic policy based on cloxacillin. The two wards, a 90-bed orthopaedic unit and a 60-bed trauma unit, had an incidence of MRSA that has remained below the hospital average (23% in 1989, 32% in 1992). Before introduction of the policy the incidence of MRSA in 1977-1979 in the orthopaedic ward was 31%, and in the trauma ward 33%. In 1989 an investigation revealed no MRSA carriers in staff of either ward. In contrast, seven MRSA carriers were found among staff and patients of three other surgical units selected, because the percentage of MRSA isolated was above the average in our hospital. However, a different type of patient is found in these units, the treatment techniques differ and broader-spectrum antibiotics are used. In addition to the usual precautions regarding nursing care and isolation techniques, the best means of reducing MRSA epidemics is to reduce the reservoir of carriers. The fall in the MRSA infection rate in orthopaedic and traumatology wards can be explained by antibiotic policy but also by other infection control measures.

Plasmid-borne cadmium resistance genes in Listeria monocytogenes are similar to cadA and cadC of Staphylococcus aureus and are induced by cadmium.

pLm74 is the smallest known plasmid in Listeria monocytogenes. It confers resistance to the toxic divalent cation cadmium. It contains a 3.1-kb EcoRI fragment which hybridizes with the cadAC genes of plasmid pI258 of Staphylococcus aureus. When introduced into cadmium-sensitive L. monocytogenes or Bacillus subtilis strains, this fragment conferred cadmium resistance. The DNA sequence of the 3.1-kb EcoRI fragment contains two open reading frames, cadA and cadC. The deduced amino acid sequences are similar to those of the cad operon of plasmid pI258 of S. aureus, known to prevent accumulation of Cd2+ in the bacteria by an ATPase efflux mechanism. The cadmium resistance determinant of L. monocytogenes does not confer zinc resistance, in contrast to the cadAC determinant of S. aureus, suggesting that the two resistance mechanisms are slightly different. Slot blot DNA-RNA hybridization analysis showed cadmium-inducible synthesis of L. monocytogenes cadAC RNA.

Plasmid-borne cadmium resistance genes in Listeria monocytogenes are present on Tn5422, a novel transposon closely related to Tn917.

The complete (6,449-bp) nucleotide sequence of the first-described natural transposon of Listeria monocytogenes, designated Tn5422, was determined. Tn5422 is a transposon of the Tn3 family delineated by imperfect inverted repeats (IRs) of 40 bp. It contains two genes which confer cadmium resistance (M. Lebrun, A. Audurier, and P. Cossart, J. Bacteriol. 176:3040-3048, 1994) and two open reading frames that encode a transposase (TnpA) and a resolvase (TnpR) of 971 and 184 amino acids, respectively. The cadmium resistance genes and the transposition genes are transcribed in opposite directions and are separated by a putative recombination site (res). The structural elements presumed to be involved in transposition of Tn5422 (IRs, transposase, resolvase, and res) are very similar to those of Tn917, suggesting a common origin. The transposition genes were not induced by cadmium. Analysis of sequences surrounding Tn5422 in nine different plasmids of L. monocytogenes indicated that Tn5422 is a functional transposon, capable of intramolecular replicative transposition, generating deletions. This transposition process is probably the reason for the size diversity of the L. monocytogenes plasmids. Restriction analysis and Southern hybridization revealed the presence of Tn5422 in all the plasmid-mediated cadmium-resistant L. monocytogenes strains tested but not in strains encoding cadmium resistance on the chromosome.

[Brucellosis among occupationally exposed people: evaluation of immunological tests after vaccination]. / Brucellose en milieu professionnel exposé: évaluation des tests immunologiques après vaccination.

For the evaluation of immunological tests during an epidemiological survey and of vaccination with the PI brucellin vaccine, in an occupationally exposed environment, a sample group of 354 subjects was studied. The vaccinal strategy was based on the outcome of a skin test for hypersensitivity: the PS brucellin test. In this framework, the serological status and evolution of individuals with positive or negative reactions to this test were analysed. Sera were studied using the buffered antigen test, indirect fluorescence immunoassay and Wright's agglutination test, as well as by PACIA and ELISA techniques with assay of IgG, IgA and IgM antibodies. The PS test, which was pivotal in this study, was compared with the lymphoblastic transformation test. One prominent aspect of this evaluation was the establishment of conventional prognostic indices for the PS and serology. The PS is definitely shown to be a convenient, reliable tool for screening. Although it does not generate sensitivity it may modify the serological status of both positive and negative individuals.

Plasmids in Listeria monocytogenes in relation to cadmium resistance.

One hundred and seventy-three unrelated Listeria monocytogenes strains isolated from humans, animals, the environment, and food were analyzed for the presence of plasmids. Extrachromosomal DNA was found in 28% of the strains. Plasmid DNA was extracted more frequently from L. monocytogenes serogroup 1 strains (35%) than from serogroup 4 strains (15%). Among strains from food and the environment, 40% and 29%, respectively, harbored plasmids, whereas only 13% of the strains from humans and animals with listeriosis bore plasmids. We also investigated the susceptibility of 90 strains to seven antibiotics and four heavy-metal salts. No antibiotic resistance could be detected, but 95.3% of the plasmid-positive strains and only 12.7% of the plasmid-negative strains were resistant to cadmium. The plasmid-determined genetic basis of cadmium resistance was proven by conjugation between strains of L. monocytogenes and by cure of the plasmid. This is the first time that plasmids of L. monocytogenes have been shown to be associated with cadmium resistance.

[Brucella vaccination in professionally exposed subjects. Prospective study]. / Vaccination brucellique en milieu professionnel exposé. Etude prospective.

This prospective phase IV study on cohort concerns a vaccine made of the phenol-insoluble fraction of Brucella abortus biotype 1 (B19 strain). Three hundred and three professionally exposed subjects entered the study; 161 out of 182 subjects (88.5 percent) with negative response to an intradermal test for detection of previous contamination accepted to be vaccinated. Booster injections were given 18 and 36 months after vaccination. Local pain was observed after 45.2 percent of injections and moderate systemic reactions after 5 percent of injections. Seropositivity after primary vaccination reached 80 percent. The booster injection, justified by a major decrease of this rate after 18 months, gave exactly the same response of the thymo-independent type. This vaccinal schedule did not result in detectable hypersensitivity. The clinical effectiveness of the vaccine could not be evaluated accurately because of the insufficient number of subjects. The possibility of subclinical infection in vaccinated subjects calls for wider comparative studies of vaccinated versus non-vaccinated subjects.