Phage Sensitivity Patterns Research Articles

Bacteriophage-antibiotic combination therapy for multidrug-resistant Pseudomonas aeruginosa: In vitro synergy testing.

Here, we investigate the impact of phage-antibiotic combinations (PAC) on bacterial killing, resistance development and outer membrane vesicle (OMV) production in multidrug-resistant (MDR) P. aeruginosa. After screening 10 well-characterized MDR P. aeruginosa strains against three P. aeruginosa phages, representative strains, R10266 and R9316, were selected for synergy testing based on high phage sensitivity and substantial antibiotic resistance patterns, while phage EM was chosen based on host range. To understand the impact of phage-antibiotic combinations (PAC) against MDR P. aeruginosa, time-kill analyses, OMV quantification and phage/antibiotic resistance testing were performed. Phage and meropenem demonstrated synergistic activity against both MDR strains. Triple combination regimens, phage-meropenem-colistin and phage-ciprofloxacin-colistin, resulted in the greatest CFU reduction for strains R9316 (3.50 log10 CFU ml-1 ) and R10266 (4.50 log10 CFU ml-1 ) respectively. PAC resulted in regained and improved antibiotic susceptibility to ciprofloxacin (MIC 2 to 0.0625) and meropenem (MIC 32 to 16), respectively, in R9316. Phage resistance was prevented or reduced in the presence of several classes of antibiotics and OMV production was reduced in the presence of phage for both strains, which was associated with significantly improved bacterial eradication. These findings support the potential of phage-antibiotic synergy (PAS) to augment killing of MDR P. aeruginosa. Systematic in vitro and in vivo studies are needed to better understand phage interactions with antipseudomonal antibiotics, to define the role of OMV production in P. aeruginosa PAC therapy and to outline pharmacokinetic and pharmacodynamic parameters conducive to PAS. This study identifies novel bactericidal phage-antibiotic combinations capable of thwarting resistance development in MDR and XDR P. aeruginosa strains. Furthermore, phage-mediated OMV reduction is identified as a potential mechanism through which PAC potentiates bacterial killing.

The pilG gene product, required for Pseudomonas aeruginosa pilus production and twitching motility, is homologous to the enteric, single-domain response regulator CheY.

The Pseudomonas aeruginosa pilG gene, encoding a protein which is involved in pilus production, was cloned by phenotypic complementation of a unique, pilus-defective mutant of strain PAO1. This mutant, designated FA2, although resistant to the pilus-specific phage D3112 was sensitive to the pilus-specific phages B3 and F116L. In spite of the unusual phage sensitivity pattern, FA2 lacked the ability to produce functional polar pili (pil) and was incapable of twitching motility (twt). Genetic analysis revealed that the FA2 pil mutation, designated pilG1, mapped near the met-28 marker located at 20 min and was distinct from the previously described pilT mutation. This map location was confirmed by localization of a 6.2-kb EcoRI fragment that complemented FA2 on the SpeI and DpnI physical map of the P. aeruginosa PAO1 chromosome. A 700-bp region encompassing the pilG gene was sequenced, and a 405-bp open reading frame, with characteristic P. aeruginosa codon bias, was identified. The molecular weight of the protein predicted from the amino acid sequence of PilG, which was determined to be 14,717, corresponded very closely to that of a polypeptide with the apparent molecular weight of 15,000 detected after expression of pilG from the T7 promoter in Escherichia coli. Moreover, the predicted amino acid sequence of PilG showed significant homology to that of the enteric CheY protein, a single-domain response regulator. A chromosomal pilG insertion mutant, constructed by allele replacement of the wild-type gene, was not capable of pilus production or twitching motility but displayed normal flagellum-mediated motility. These results, therefore, suggest that PilG may be an important part of the signal transduction system involved in the elaboration of P. aeruginosa pili.

A cryptic plasmid of indigenous Rhizobium meliloti possesses reiterated nodC and nifE genes and undergoes DNA rearrangement

Indigenous Rhizobium meliloti were previously characterized on the basis of plasmid profiles and phage sensitivity patterns (phage types). Rhizobium meliloti 1076, which contained two cryptic plasmids, was one of four isolates comprising phage type 23. In this study, the large cryptic plasmid pVS1(size >500 b) was transferred from isolate 1076 into the plasmid-free strain of Agrobacterium tumefaciens UBAPF1. This plasmid contained nucleotide sequences homologous to genes for nodulation (nodB, nodC) and nitrogen fixation (nifH, nifD, nifK, nifE). Cosmid clones possessing the nod and nif homologous sequences, which had been selected from a genomic bank of A. tumefaciens UBAPF1 containing pVS1, complemented R. meliloti nodC and nifE mutants, respectively. These results demonstrate that the nodC and nifE homologous sequences are functionally expressed. Three of four isolates comprising phage type 23 possessed a megaplasmid band in agarose gels characteristic of R. meliloti, as well as two cryptic plasmids. The fourth isolate (No. 323) lacked the large cryptic plasmid corresponding to pVS1, but instead showed a band of lesser mobility than that of the megaplasmids. Nevertheless, its restricted genomic DNA retained the nodC and nifE hybridizing fragments characteristic of pVS1, indicating that the cryptic plasmid has undergone DNA rearrangement. Key words: Rhizobium, plasmid, reiteration, genes, rearrangement.

Comparison of ribotyping with conventional methods for the type identification of Enterobacter cloacae

An Escherichia coli rRNA probe was compared with a combination of O serotyping, phage susceptibility, and biotype pattern for the type identification of strains of Enterobacter cloacae. Forty-five isolates of E. cloacae from 36 patients in nine hospitals were examined. By conventional typing, only 26 (57.7%) could be assigned to a specific serotype and 6 (13.3%) were autoagglutinating owing to rough lipopolysaccharide antigens. All isolates could be assigned to one of three biotypes, but many phage sensitivity patterns were evident. Twenty-nine distinct strains were identified by combined typing. Probing of EcoRI and BamHI digests of chromosomal DNA with a cDNA copy of E. coli rRNA proved to be highly discriminating between strains. Thirty different ribotypes based on 28 bands were recorded. Overall, agreement between the ribotyping and combined typing methods was good (84.4%), and discrepancies were generally confined to serologically unclassifiable strains and variability in biotype codes. Ribotyping was reproducible, and five of six pairs of isolates from the same and different patients gave identical hybridization profiles on separate occasions. We conclude that ribotyping is a highly discriminatory and reproducible method for the typing of E. cloacae, but in most outbreaks it offers little increase in discrimination over traditional methods.

Observations on phage-typing of Clostridium difficile: Preliminary evaluation of a phage panel

Of the various methods now employed to type Clostridium difficile, the performance of the phage-typing, as proposed by Sell et al., was investigated on a large collection of isolates from various local epidemiological settings. Because those isolates appeared in preliminary trials mostly resistant to the reference phages, the panel was enlarged by including newly isolated phages. The new set was redundant but better suited to the purpose: out of 287 phage sensitivity patterns 8.4% only were resistant in comparison to the 67.6% resistant to the reference one.

Use of Phage Typing and Fatty Acid Analysis for the Identification of Bacilli Isolated from Food Packaging Paper and Board Machines

Summary Fourty-eight Bacillus reference strains representing 17 different species and 22 Bacillus isolates from paper and board (carton) mills were characterized by phage sensitivity and whole cell fatty acid analysis. On the basis of phage sensitivity patterns obtained with 44 phages of 10 different Bacillus species, the strains could be divided into 27 different phage types. Whole cell fatty acid profiles allowed classification of strains into 20 different fatty acid biovars. These two approaches together allowed the identification of 19 out of the 22 paper and board mill isolates in agreement with results obtained by classical identification methods based on the biochemical and physiological properties.

The use of phage-sensitivity patterns for tracing heat labile toxin-positive (LT+) Escherichia coli.

The heat-labile toxin (LT) positive Escherichia coli colonies from 785 stool specimens obtained during a cholera vaccine trial were examined for their phage-sensitivity pattern to 31 E. coli phages. These specimens originated from 105 index cases and their contacts. Isolates with common phage-sensitivity patterns were grouped and were studied further both serologically and for their plasmid profile. The largest group (42 isolates) belonged to serogroup O78 and the second largest group (19 isolates) belonged to serogroup O6. There were 23 index cases which had E. coli with the same phage-sensitivity pattern as some of their contact strains. The identity of isolates from 16 index cases with strains from their respective contacts could be verified serologically. For the remaining seven index cases and their contacts, the isolates did not agglutinate with available antisera. However, subsequent studies demonstrated that, when the phage-sensitivity pattern among the strains matched, the plasmid profiles of these strains also matched. This further indicates the ability of phage-sensitivity patterns to serve as markers in tracing strains.

Taxonomic Revision of the Genus Saccharomonospora and Description of Saccharomonospora glauca sp. nov.

Representatives of Saccharomonospora viridis (type strain DSM 43017), Saccharomonospora caesia (type strain DSM 43044), and Saccharomonospora internatus (type strain DSM 43671), together with 52 Saccharomonospora isolates from compost, manure, hay, and soil, were characterized by determining morphological, biochemical, and physiological properties, phage sensitivity, antibiotic activity, enzyme and protein patterns (polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and restriction patterns of chromosomal deoxyribonucleic acids. The type strain of S. internatus proved to be identical to S. viridis, and thus S. internatus should be regarded as a synonym of S. viridis. A total of 16 strains differed significantly from S. viridis and S. caesia and are proposed as members of a new species, Saccharomonospora glauca (type strain, DSM 43769). Since the three species differ in phage sensitivity, production of antibiotic substances, and enzyme, protein, and deoxyribonucleic acid restriction patterns, these properties can be used for reliable species identification.

β-Lactamase-Producing Mutants of Streptococcus cremoris

Penicillin-resistant mutants were isolated for six strains of Streptococcus cremoris used in commercial Cheddar cheese manufacture after treatment with N-methyl-N-nitro-N-nitrosoguanidine. The resistant mutants had an elevated minimal growth inhibitory concentration, 2.5μg (4.13 units)/ml, for penicillin G and other β-lactam antibiotics as compared with the penicillin-susceptible parent strains, which were each sensitive to .05μg (.08 units)/ml. Penicillin resistance was due to the production of β-lactamase. Plasmid DNA was not demonstrated in partially purified lysates of four mutants. Mutants had normal cellular morphology but altered phage sensitivity patterns. All except one strain were able to support complete phage adsorption. Resistance was retained after 20 passages in absence of penicillin.

Effect of rifampin resistance on nodulating competitiveness of Rhizobium meliloti

An assessment was made of the effect of high concentration rifampin resistance on the nodulating competitiveness of five strains of Rhizobium meliloti. The results indicate that the acquisition of rifampin resistance by R. meliloti is generally associated with a significant loss of nodulating competitiveness and an altered RNA polymerase insensitive to the action of rifampin. All mutants were similar to their parent strains with respect to growth rates, phage sensitivity patterns, and symbiotic effectiveness. The data suggest that rifampin resistance in R. meliloti is unsuitable as a marker for ecological studies.

Brucella phagotypes: their relation to the spread of infection in Italy.

The spread of Brucella infection in man in Italy during the last three years is briefly reviewed with reference to the Brucella strains collected and typed. The strains were tested by H2S production, dye-inhibition, agglutination by monospecific sera, and sensitivity to a phage set. From 108 tested strains, 91 have been identified by means of the traditional tests as Br. melitensis, sometimes with some irregularities, 10 as Br. abortus, while 7 were not typable. It was possible to recommend some phage sensitivity patterns of Br. melitensis for epidemiological purposes: one phagotype is much more frequent in the north, another in the south of Italy. However, the currently recommended phagotypes must be considered provisional and will be modified in the light of more extensive studies.

Temperature-sensitive mutants in rfaI and rfaJ, genes for galactosyltransferase I and glucosyltransferase II, for synthesis of lipopolysaccharide in Salmonella typhimurium

Certain rough mutants of Salmonella typhimurium LT2 were shown to be temperature sensitive for the production of lipopolysaccharide (LPS). When grown at the restrictive temperature (42 or 45 degrees C), the cells contained LPS deficient in O (somatic) side chains, based on phage-sensitivity data and gel electrophoresis of the LPS. Cells grown at the permissive temperature, 30 degrees C, made LPS resembling that of smooth cells. The mobility of the LPS in gels, the phage sensitivity patterns, and gas chromatographic analysis indicate that LPS of 45 degrees C-grown cells of SA126 (rfaJ3012) is of chemotype Rb2, with one glucose and two galactose units (and thus inferred to be due to a mutation in rfaJ), and LPS of 45 degrees C-grown cells of SA134 (rfa13020) is of chemotype Rb3, with one glucose and one galactose unit (inferred to be rfaI). These inferences were confirmed, for pKZ26 (pBR322-rfaGBIJ) and pKZ27 (pBR322-rfaGBI) both complement rfaI3020, but only pKZ26 complemented rfaJ3012. In addition, pKZ26 carrying a Tn5 insertion resulting in loss of complementation of a known rfaJ mutation, but not of rfaG, B, or I, also resulted in loss of rfaJ3012 complementation. Based on gel analysis, there is a small amount of the LPS containing smooth side chains in cells of SA126 grown at 45 degrees C; following a switch to 30 degrees C, the amount of LPS with O side chains gradually increased, and the amount of core LPS was reduced, though even after 3 h the LPS does not fully resemble that of smooth strains.(ABSTRACT TRUNCATED AT 250 WORDS)

New Salmonella typhimurium mutants with altered outer membrane permeability.

We describe three new classes of Salmonella typhimurium mutants with increased sensitivity to hydrophobic agents. In contrast to many previously described mutants, the phage sensitivity pattern of these mutants did not give any indication of defective lipopolysaccharide. Furthermore, they had no detectable changes in their phospholipid or outer membrane protein composition, and their growth rate and cell morphology were normal. Class B mutants were nearly as sensitive to novobiocin, fusidic acid, erythromycin, rifampin, and clindamycin as are deep rough (heptoseless) mutants; in addition they were sensitive to methicillin, penicillin (to which heptoseless mutants are resistant), gentian violet, and anionic and cationic detergents. Class A and C mutants had less sensitive, but characteristic phenotypes. None of the three classes were sensitive to serum bactericidal action. The class B mutation mapped between map positions 7 and 11 on the S. typhimurium chromosome, and the class C mutation mapped between positions 5 and 7. The map position for the class A mutation remained undefined, but it was separate from the class B and C mutations and, like those, did not correspond to any gene loci known to participate in the synthesis of major outer membrane constituents.

The effect of acquired resistance to mercury on the phage sensitivity pattern ofStaphylococcus aureusand its adherence to buccal epithelial cells

The study on the effect of acquired resistance to mercury on the phage sensitivity pattern of Staphylococcus aureus and its adherence to buccal epithelial cells showed increased lag period in growth and inverse relationship between the mercury resistance and cell wall teichoic acid, protein A and binding of cells to normal humal buccal cells. The phage pattern was found to change with the development of mercury resistance.

The effect of acquired resistance to mercury on the phage sensitivity pattern of Staphylococcus aureus and its adherence to buccal epithelial cells

The effect of acquired resistance to mercury on the phage sensitivity pattern of Staphylococcus aureus and its adherence to buccal epithelial cells

Bacteriophage and bacteriocin typing scheme for Clostridium difficile.

The study of the epidemiology of infection with Clostridium difficile would be aided by a way to type individual bacterial isolates. We therefore sought bacteriophages for use in typing. With mitomycin C exposure (3 micrograms/ml), filtrates from 10 strains of C. difficile had plaque-forming lytic activity on other C. difficile strains. Individual phage were passaged and made into high-titer stock preparations for typing. Electron microscopy revealed tailed phage particles from one such preparation. In addition to phage, inhibitory activity without distinct plaque formation consistent with bacteriocins was observed for 20 strains. C. difficile isolates from 16 patients taken 1 to 14 days apart were similar in their phage sensitivity pattern, whereas isolates from separate geographic locations showed a great variety of patterns. We conclude that bacteriophage should be useful for typing strains of C. difficile.

Bacteriophage sensitivity patterns among bacteria isolated from marine waters

Phage-host cross-reaction tests were performed with 774 bacterial strains and 298 bacteriophages. The bacteria (bacteriophages) were isolated at different times from water samples collected in the Atlantic Ocean between the European continental shelf and the Sargasso Sea: 733 (258) strains; in the North Sea near Helgoland: 31 (31) strains; and in the Bay of Biscay: 10 (9) strains. Of the Atlantic Ocean bacteria 326 were found to be susceptible to one or more Atlantic Ocean bacteriophage(s). The bacteriophage sensitivity patterns of these bacteria vary considerably, placing 225 of them in two large clusters of bacteriophage-host systems. Taking all into account, 250 of the 326 Atlantic Ocean bacteria are different from each other. This high degree of variation among the bacteria distinguishes microbial populations derived from widely separated and regions of the Atlantic Ocean. It also sets apart from each other the populations derived from samples collected at successive stations some 200 miles apart, although to a lesser degree. With bacterial populations found from samples collected on the way back and forth between Europe and the Sargasso Sea a gradual change was observed from western phage sensitivity patterns to eastern ones. Sixty-nine Atlantic Ocean bacteria are sensitive to bacteriophages isolated from the North Sea and the Bay of Biscay; of these only 26 strains are also susceptible to Atlantic Ocean phages. The interpretation of the results is based on the hydrographical conditions prevailing in the northern Atlantic Ocean including the North Sea, and on the assumption that the microbial populations investigated have undergone genetic changes while being transported within water masses from west to east.

Mapping of genes determining penicillin-resistance and serum-sensitivity in Salmonella enteritidis.

Two presumptive single-step mutants, resistant to penicillin and sensitive to the bactericidal effect of normal human serum, were isolated on penicillin gradient plates from a smooth, penicillin-sensitive, serum-resistant strain of Salmonella enteritidis (O9,12; gal hisEI cys). The chemical composition of their lipopolysaccharides, their phage-sensitivity patterns, and their serological and cultural properties showed one to be 'part-rough' and the other smooth. Hfr strains with O antigens O4,5,12 or O1,4,5,12 (two Salmonella typhimurium and one S. abony) and one S. enteritidis F', O9,12, were crossed with the S. enterititis O9,12 mutants. The results with the part-rough mutant indicate that its penicillin-resistance, serum-sensitivity and rough phage pattern result from a single mutation between hisEI and the part of the rfb gene cluster determining O specificity, 4 (abequose) or 9 (tyvelose). Transduction experiments confirmed that the mutation is closely linked to the his operon. This mutation is inferred to cause an incomplete defect in a transferase for galactose, mannose or rhamnose, the smooth sugars common to O4,5,12 and O9,12. Results from similar crosses to the smooth, serum-sensitive, penicillin-resistant S. enteritidis mutant indicate that its serum-sensitivity is not linked to his. The occasional independent segregation of penicillin-resistance and serum-sensitivity suggests that other loci modify penicillin-resistance.

Lipopolysaccharide Core Defects in Salmonella typhimurium Mutants Which Are Resistant to Felix O Phage but Retain Smooth Character

FOR mutants of Salmonella typhimurium are resistant to Felix O phage, whose receptor includes the N-acetylglucosamine branch of the lipopolysaccharide (LPS) core, but smooth in cultural properties, antigenic character and phage sensitivity pattern (MacPhee et al., 1975). The rfa(FOR) genes determining the FOR character of nine mutants were transduced into a smooth cysE pyrE recipient: the nine FOR transductants (and a tenth FOR mutant) were then made rfb (i.e. unable to make O chains) by transduction or Hfr crosses. The rfb FOR strains were sensitive to FO phage but nearly all of them showed a somewhat reduced efficiency of plating and diminished rate of adsorption of the phage. This observation and the Ra (complete core) serological activity of their LPS (tested by haemagglutination inhibition) indicate the presence of some, but less than the normal number of, completed core chains in FOR rfb LPS. On the basis of the sensitivities of the FOR transductants and their rfb derivatives to various 'rough-specific' phages, their increased sensitivities to some antibiotics and to deoxycholate and the serological activity of the rfb FOR LPS in various incomplete core systems, the mutants were divided into three groups: (i) five mutants with probable defects in previously undetected rfa gene(s) concerned with formation of both the galactose I and the galactose II units of the LPS core; (ii) two mutants with defects inferred to affect the structure of the inner part of the core and also interfere with addition of the N-acetylglucosamine branch; (iii) three mutants in which no type of incomplete core could be detected, probably affected in formation of the inner part of the core chain. The mutation of one mutant of the last class, unlike those of the other nine mutants tested, lay outside the cysE-pyrE segment, in the 90 to 116 min region of the linkage map.

Epidemiological markers for pseudomonas aeruginosa.

A non-lysogenic strain of Pseudomonas aeruginosa maintained its phage sensitivity pattern and serogroup specificity unchanged for 10 weeks in ex-germfree, mono-contaminated rats before infection with phage. After infection with phage, phage conversions of serogroup specificity and lysotype were observed. With the same combination of bacterial and phage strains, the same serogroup was obtained in vitro and in vivo. In vitro conversion occurred also to serogroups that were not detected in vivo. Upon lyophilization, converted bacterial clones from the in vivo experiment lost their phage and simultaneously reverted to the original phage type and serogroup. These findings may have implications for the understanding of the degree of stability in epidemiological typing results for P. aeruginosa.