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.     

Isolation, purification and ultrastructure of bacteriophages from natural mosquito breeding places in Egypt

Two bacteriophages were isolated from field collected samples representing two different mosquito breeding places. The phage AB-1 (isolated from Abheit village, Faiyoum Governorate "seepage water") and the phage GA-2 (isolated from El-Gabal El-Asfer Qualyobia Governorate "sewage drain water") were purified. Both bacteriophages were ultrastructurally described with respect to their morphology, dimensions, phases of bacterial attack and lysogeny. No major differences were observed between both isolated phages in relation to specificity, however; they were isolated from two different types of breeding places and two different geographic areas as well. This study may assume a wide host range of the isolated phages and reflect how bacterial insecticides used for mosquito larval control could be inhibited by such bacteriophage.     

Bacteriophage diversity in the North Sea

In recent years interest in bacteriophages in aquatic environments has increased. Electron microscopy studies have revealed high numbers of phage particles (10(4) to 10(7) particles per ml) in the marine environment. However, the ecological role of these bacteriophages is still unknown, and the role of the phages in the control of bacterioplankton by lysis and the potential for gene transfer are disputed. Even the basic questions of the genetic relationships of the phages and the diversity of phage-host systems in aquatic environments have not been answered. We investigated the diversity of 22 phage-host systems after 85 phages were collected at one station near a German island, Helgoland, located in the North Sea. The relationships among the phages were determined by electron microscopy, DNA-DNA hybridization, and host range studies. On the basis of morphology, 11 phages were assigned to the virus family Myoviridae, 7 phages were assigned to the family Siphoviridae, and 4 phages were assigned to the family Podoviridae. DNA-DNA hybridization confirmed that there was no DNA homology between phages belonging to different families. We found that the 22 marine bacteriophages belonged to 13 different species. The host bacteria were differentiated by morphological and physiological tests and by 16S ribosomal DNA sequencing. All of the bacteria were gram negative, facultatively anaerobic, motile, and coccoid. The 16S rRNA sequences of the bacteria exhibited high levels of similarity (98 to 99%) with the sequences of organisms belonging to the genus Pseudoalteromonas, which belongs to the gamma subdivision of the class Proteobacteria.     

Characteristics of Staphylococcus aureus strains isolated from clinical and non-clinical human sources in Trinidad: susceptibility to bacteriophages and antimicrobial agents, and toxigenicity

The susceptibility of Staphylococcus aureus strains isolated from human clinical and non-clinical sources in Trinidad to bacteriophages and antimicrobial agents was determined. The ability of the strains to produce enterotoxins and toxic shock syndrome toxin-1 (TSST-1) was also investigated. Of the 554 strains tested, 454 (81.8%) were susceptible to international phage set (IPS) phages with strains isolated from bacteruria (57.1%) and bacteremia (53.3%) having a low sensitivity compared to isolates from aspirates (87.3%) and anterior nares (97.4%). All sources combined, strains were most susceptible to phages belonging to several groups (mixed). Overall, 419 (75.6%) strains were resistant to one or more of nine antimicrobial agents tested. Resistance to penicillin was most prevalent, with 413 (74.5%) strains found to be resistant. Prevalence of resistance to tetracycline, gentamicin, oxacillin, cefuroxime and ciprofloxacin was 5.1%, 2.0%, 0.7%, 0.4% and 0.4%, respectively. Of the 554 strains tested, 307 (55.4%) produced staphylococcal enterotoxins A (SEA), B (SEB), C (SEC) and D (SED) singly or in combination. Strains recovered from high vaginal swabs were least enterotoxigenic (40.0%) as compared to umbilical infection isolates which were most enterotoxigenic (78.9%). TSST-1 was produced by 95 (19.0%) out of 499 strains tested, with isolates from bacteruria found to be most toxigenic (33.3%). It was concluded that the S. aureus strains tested were highly susceptible to bacteriophages and antimicrobial agents (except penicillin) and that enterotoxigenic and TSST-1 producers were widespread and have an aetiologic potential.     

Vibrio cholerae O1 in fecal samples from the urban population of Manacapuru, AM

The study was carried out to identify asymptomatic carriers of V. cholerae O1 in Manacapuru, AM. 1249 feces samples was obtained by rectal swab and cultivated. Had no growth of V. cholerae. On the other hand were isolated and identified: V. furnissii in 12 (0.9%) samples, V. fluvialis in 4 (0.3%) and V. hollisae in 1 (0.1%).     

Dextropropoxyphene deaths in Denmark from the health authority point of view

Using electron microscopy and DNA-DNA-hybridization, 113 virulent and temperate bacteriophages specific for P. aeruginosa have been assigned to 23 species. In most cases, especially in virulent phages, both particle morphology and DNA homology types were in good correlation and their use was sufficient for clear-cut definition of phage species. No virulent phages of different species had any DNA homology. DNA homology was detected between temperate phages of several species. Temperate phages formed two large groups of two and seven species, respectively. The first group included all transposable bacteriophages. The extent of interspecies DNA homology of phages belonging to each group was not more than 10-15% (except for 25% for phages D 3 and KF 1). No DNA homology was between phages of different groups. The possible origin and function of homologous sequences (genetic modules, linkers, occasional insertional sequences) are discussed. One of the phages (phi C 15) may be considered as the result of recombination between phages belonging to two different species, 295 and SM.     

Selection and properties of totally phage-resistant mutant Pseudomonas putida PpG1

The efficiency of using bacteria in open systems to degrade different anthropogenic toxic pollutants can depend strongly on the interaction between these bacteria and natural bacteriophages. The possibility of selecting bacterial Pseudomonas putida mutants resistant to all bacteriophages of this species known so far was tested (in our work, these mutants were designated totally phage-resistant mutants). In a model experiment, changes in the composition of a population upon prolonged growth of bacteria in the presence of one of the virulent phages were examined. On the basis of the results obtained, it is postulated that: (1) Mutants differing in resistance to various phages accumulate in a population; relative numbers of different mutants can undergo alterations over the course of time; mutants selected in the presence of a given virulent phage do not often manifest complete resistance to this phage. (2) It is possible to isolate totally phage-resistant mutants of P. putida PpG1. These mutants carry up to three different mutations simultaneously; however, these mutants regain sensitivity to many phages upon pseudoreversion occurrence.     

Abundance in sewage of bacteriophages that infect Escherichia coli O157:H7 and that carry the Shiga toxin 2 gene

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7, but data on the occurrence and distribution of such phages as free particles in nature were not available. An experimental approach has been developed to detect the presence of the Shiga toxin 2 (Stx 2)-encoding bacteriophages in sewage. The Stx 2 gene was amplified by PCR from phages concentrated from 10-ml samples of sewage. Moreover, the phages carrying the Stx 2 gene were detected in supernatants from bacteriophage enrichment cultures by using an Stx 2-negative E. coli O157:H7 strain infected with phages purified from volumes of sewage as small as 0.02 ml. Additionally, the A subunit of Stx 2 was detected in the supernatants of the bacteriophage enrichment cultures, which also showed cytotoxic activity for Vero cells. By enrichment of phages concentrated from different volumes of sewage and applying the most-probable-number technique, it was estimated that the number of phages infectious for E. coli O157:H7 and carrying the Stx 2 gene was in the range of 1 to 10 per ml of sewage from two different origins. These values were approximately 1% of all phages infecting E. coli O157:H7.     

Isolation of Enterobacteriaceae bacteriophage particles catalysing cell wall lipopolysaccharide degradation

Using pairs of smooth and rough forms of Enterobacteriaceae, six smooth-specific bacteriophages were isolated from sewage and another was obtained from Dr Hedda Milch, Budapest. Upon incubation of the individual extracted (smooth) host cell wall lipopolysaccharides with the homologous purified viruses, liberation of reducing groups (i.e. of about di- to nonasaccharides) was observed in four cases, indicating the action of glycanases - but no liberation of acetic acid, indicating the absence of esterase activity. Under the electron microscope, all phages were seen to exhibit Bradley group B or C morphology and to carry tail spikes.     

Potentially pathogenic vibrios associated with mussels from a tropical region on the Atlantic coast of Brazil

Mussels (Perna perna) harvested on the coast of Ubatuba, in three different stations in the State of S?o Paulo, Brazil, were examined for Vibrio spp. over a 1 year period. The ranges of most probable number (MPN 100 g-1) were: Vibrio alginolyticus (< 3-24,000), V. parahaemolyticus (< 3-24,000), V. fluvialis (< 3-1100), V. cholerae non-O1 (< 3-23), V. furnissii (< 3-30), V. mimicus (< 3-9) and V. vulnificus (< 3-3). The highest incidence was observed for V. alginolyticus (92-100%), followed by V. parahaemolyticus (67-92%), V. fluvialis (34-67%), V. vulnificus (8-17%), V. furnissii (0-17%), V. mimicus (0-17%) and V. cholerae non-O1 (0-8%). Tests for virulence factors were positive in 34.1% of the vibrios in the rabbit ileal loop and 31.7% in the Dean test. Positive results in the Kanagawa test were obtained with 0.51% of V. parahaemolyticus strains. The mean values (MPN 100 g-1) of faecal coliforms in mussels from the three regions varied from 1100 to 44,000, and seawater collected at the same stations gave average values for faecal coliforms in the range 18-3300 MPN 100 ml-1. These results highlight the potential risks of food poisoning associated with raw or undercooked seafood.     

Isolation and properties of Escherichia coli mutants which are nonpermissive for the growth of phage lambda

By selecting survivors of lambda phage infection, mutants of Escherichia coli K12 that block reproduction cycle of the phage have been isolated. Fourteen of these phage-tolerant mutants (lam mutants) were chosen and characterized biochemically and genetically. It was shown that these mutants were tolerant to infection by all the lambdoid phages, except for few cases, but they were susceptible to infection by a non-lambdoid temperate phage (phi299), P1 or T phages. The mutants can be divided into at least three groups: (1) A mutant (lam 16) strain that seems to block normal penetration of phage DNA: (2) Three mutant (lam 64, lam 67 and lam 71) strains that block an "early" step(s) of phage growth, including phage DNA synthesis: (3) Six mutant (lam 24, lam 25, lam 26, lam 27, lam 646 and lam 6) strains that block normal functioning of the gene E products and produce unusual head structures. Some lambdoid phages and lambda mutants that overcome the interference by the lam mutations have been obtained, and were used as tools for characterizing the host mutations. Two (lam 12 and lam 13) mutant strains and one (lam 1) mutant were inferred as affecting the expression of "late" genes, and early gene, respectively, by this test.     

Diarrhea associated with Vibrio fluvialis in the United States

We report the isolation in the United States of Vibrio fluvialis from the stools of a patient who had severe watery diarrhea without fever and who subsequently died. V. fluvialis, a known enteric pathogen in other parts of the world, should be suspected in patients with watery diarrhea, especially in coastal areas.     

Lysogeny in Pasteurella multocida]

Studied was the possibility of lysis-producing factors (the phenomenon of lysogeny) with 59 strains of Pastuerella multocida isolated in Bulgaria, Cuba, and Czechoslovakia. It was found that eleven of them were lysogenic in terms that a total of 12 bacteriophage strains were isolated from them; one of them yielded 2 phages. Established were three different indicator strains of Pasteurella multocida-97, SHD, and R-II--by means of which 3 different groups of P. multocida phages were isolated. The latter were stabilized and allowed to multiply up to 10(11) phage particles per 1 cc through continuous passaging, and they could be be stored at + 4 degrees C. In accordance with the host strain for multiplying the isolated P. multocida phages were divided into three different groups: phages 3, 4, 5, 6, 22, and Sl fell into group II, and phages 1075 and S-2--to group III.     

Comparison of ribotyping and randomly amplified polymorphic DNA PCR for characterization of Vibrio vulnificus

A total of 85 isolates of Vibrio vulnificus were characterized by ribotyping with a probe complementary to 16S and 23S rRNA of Escherichia coli and by randomly amplified polymorphic DNA-PCR (RAPD-PCR) with a 10-mer oligonucleotide primer. The RAPD-PCR results were scanned, and the images were analyzed with a computer program. Ribotype membranes were evaluated visually. Both the ribotyping and the RAPD-PCR results showed that the collection of strains was genetically very heterogeneous. Ribotyping enabled us to differentiate U.S. and Danish strains and V. vulnificus biotypes 1 and 2, while the RAPD-PCR technique was not able to correlate isolates with sources or to differentiate the two biotypes, suggesting that ribotyping is useful for typing V. vulnificus strains whereas RAPD-PCR profiles may subdivide ribotypes. Two Danish clinical biotype 2 strains isolated from fishermen who contracted the infection cleaning eels belonged to the same ribotype as three eel strains (biotype 2), providing further evidence that V. vulnificus biotype 2 is an opportunistic pathogen for humans. One isolate (biotype 2) from Danish coastal waters also showed the same ribotype as the eel strains. This is, to our knowledge, the first time the isolation of V. vulnificus biotype 2 from coastal waters has been described.     

Analysis of an outbreak of non-phage-typeable methicillin-resistant Staphylococcus aureus by using a randomly amplified polymorphic DNA assay

A cluster of methicillin-resistant Staphylococcus aureus (MRSA) infections among patients on an intensive care unit (ICU) was detected by routine infection control surveillance. In the period from 5 January to 22 June 1995, 10 patients on the ICU and a further 6 patients (5 on one ward that had received colonized patients transferred from the ICU) were affected by MRSA strains with the same antibiotic susceptibility patterns. Seven (44%) of these 16 colonized patients developed MRSA bacteremia. MRSA isolates with the same characteristics were also found on the hands of one member of the ICU staff. The isolates were untypeable by phage typing, but 15 of 17 outbreak strains analyzed genetically had identical randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) profiles. A single strain of MRSA that was nontypeable by phage typing and that was isolated on the ICU on 1 January and six nontypeable and epidemiologically unrelated MRSA isolates all had RAPD profiles distinct from that of the outbreak strain. Implementation of strict infection control measures stopped the further spread of MRSA on the ICU, the affected general ward, and seven other wards that received MRSA carriers from the ICU. Although nontypeable by phage typing and not previously recognized as an epidemic strain, this strain of MRSA was readily transmissible and highly virulent. RAPD typing was found to be a simple, rapid, and effective method for the epidemiological investigation of this outbreak, and performance of typing by this method was simpler and less time-consuming than that of typing by PFGE. RAPD typing may have more general application for the study of S. aureus infections in hospitals.     

Eva Klein: the Ruggero Ceppellini lecturer, 1997

We report a case of Vibrio fluvialis gastroenteritis in an infants 3 1/2 weeks old. The case was unusual because no likely epidemiologic risk factors were involved. Since several other such cases in young infants have been reported, V fluvialis should be considered in the differential diagnosis of infantile gastroenteritis.     

Biotyping of Campylobacter strains isolated in Lagos, Nigeria using the modified Preston biotype

Fifty-eight Compylobacter strains were isolated from children with diarrhoea at various health centres in Lagos and from healthy chicken. Twenty-nine strains of Campylobacter were isolated from humans, while the same number were isolated from chicken. The strains were biotyped using the modified Preston biotype scheme. The Preston biotyping results have been compared with the results of Penner serotyping. Out of fifty-eight strains studied, the technique identified ten strains (17%) as C. coli, three (5%) as C. lari and fourty-five (78%) as C. jejuni, by the coding system. This technique identified twenty-eight Campylobacter species. This method highlights the usefulness of this technique in the biotyping of local strains, however, when the two schemes are used in combination they give excellent typing results suitable for epidemiological purposes.