Penneri Strains Research Articles

ISOLATION, CHARACTERIZATION AND BIOCIDAL STUDIES OF PREDOMINANT BIOFOULING PROTEUS STRAINS AND FUNGI ON BOAT HULLS

Coliform bacteria occurring in bio?lms were isolated from the hulls of boats at the Ojo water front, Lagos Nigeria using MacConkey agar and Eosine methylene blue agar. They were subsequently identi?ed by phylogenetic analysis using 16S rDNA sequences and assessed for antimicrobial susceptibility to industrial in-can biocide (Acticide BX-H). All the sequences were classified using the Ribosomal database project (RDP) and submitted to the National Center for Biotechnology Information. Analysis using 16S rDNA indicated that the 16 strains sequenced belonged to the Enterobacteriaceae family and included Proteus mirabilis strain BB2000, P. mirabilis strain UT5, P. vulgaris strain CYPV1, P. penneri strain YCY34 and Proteus sp.CIFE_HT14. Fungi isolated were Fusarium oxysporum, Aspergillus flavus, A. niger, Candida albicans and Penicillim chrysogenum. The minimum inhibitory concentration (MIC) observed was 20µL/ml for P. mirabilis, P. vulgaris and P. penneri strains respectively. For fungal isolates, MICs were 40µL/ml, 40µL/ml, 20µL/ml, 40µL/ml and 40µL/ml for F. oxysporum, A. flavus, A. niger, C. albicans and P. chrysogenum respectively. Zones of inhibition recorded were 20.25mm, 17.75mm, 15.50mm, 15. 22mm and 14.50mm for P. penneri strain YCY34, P. vulgaris strain CYPV1, P. mirabilis strain BB2000, P. mirabilis strain UT5 and Proteus sp. CIFE_HT14 respectively. Result of the study showed that the biocide has a broad spectrum antimicrobial activity.

The unique structure of bacterial polysaccharides - Immunochemical studies on the O-antigen of Proteus penneri 4034-85 clinical strain classified into a new O83 Proteus serogroup

The serological classification scheme of the opportunistic Proteus bacilli includes a number of Proteus penneri strains. The tested P. penneri 4034-85 strain turned out to be serologically distinguished in ELISA and Western blotting. The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of this strain and studied by sugar and methylation analyses and dephosphorylation along with 1H and 13C NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, HMBC, and HSQC-TOCSY experiments, The O-polysaccharide was found to have a linear repeating unit containing glycerol 1-phosphate and two residues each of Gal and GlcNAc. The following O-polysaccharide structure was established, which, to our knowledge, is unique among known bacterial polysaccharide structures:→4)-β-d-GlcpNAc-(1→3)-α-d-Galp-(1→3)-β-d-GlcpNAc-(1→2)-β-d-Galp-(1→3)-Gro-1-P-(O→.

Classification of a Proteus penneri clinical isolate with a unique O-antigen structure to a new Proteus serogroup, O80

Proteus penneri is an opportunistic pathogen, which may cause severe diseases, most frequently urinary tract infections in immunocompromised patients. P. penneri Br 114 exhibiting a good swarming growth ability as an S-form strain was isolated from a wound of a patient in Łódź, Poland. Serological studies using ELISA and Western blotting and chemical analyses along with 1H and 13C NMR spectroscopy showed that the O-antigen (O-polysaccharide) of this strain is unique among the known Proteus serotypes O1–O79. It possesses a linear pentasaccharide repeating unit containing a partially O-acetylated amide of d-glucuronic acid (GlcA) with l-serine having the following structure:▪These data are a basis for creating a new Proteus serogroup, O80, so far represented by the single Br 114 isolate. The O80 is the 21st O-serogroup containing P. penneri strains and the fourth serogroup based on Proteus spp. clinical isolates from Łódź, Poland.

Identification of a Proteus penneri isolate as the causal agent of red body disease of the cultured white shrimp Penaeus vannamei and its control with Bdellovibrio bacteriovorus

Bacteriosis has become a major economic problem in the farming of the Pacific white shrimp Penaeus vannamei. However, no definitive data are available about Proteus penneri infection in cultured P. vannamei and its control. In this study, a virulent strain NC was isolated from diseased P. vannamei suffering from red body disease and identified as a P. penneri isolate through phylogenetic analysis and ATB 32GN system. A phylogenetic constructed tree using the neighbour-joining method identified the NC isolate as a P. penneri strain. In addition, Bdellovibrio bacteriovorus conferred significant protection against P. penneri: it exhibited significant bacteriolytic effects on the pathogenic P. penneri, had a wide prey range towards Proteus pathogens, and displayed a good protective efficacy on experimental P. penneri infection in P. vannamei. To the best of our knowledge, this is the first report of farmed P. vannamei infected with P. penneri and its control with B. bacteriovorus.

Serological Studies of Proteus penneri Strains Determining Qualification to Appropriate O-serogroup

Our Department of General Microbiology created a wide collection of P. penneri isolates and classified most of them into 19 different O-serogroups. This work describes the classification of 12 remaining P. penneri strains. The lipopolysaccharides extracted from P. penneri strains were tested in an enzyme-linked immunosorbent assay (ELISA) with selected O-antisera against Proteus sp. strains. Homologous and cross-reacting systems were checked in: passive immunohemolysis (PIH), inhibition of ELISA and PIH and Western blot procedure. These studies led to the qualification of tested P. penneri strains to five Proteus sp. O-serogroups, thus completing the serological classification of the whole collection.

16S-23S rDNA internal transcribed spacer regions in four Proteus species

Proteus is a Gram-negative, facultative anaerobic bacterium. In this study, 813 Proteus 16S-23S rDNA internal transcribed spacer (ITS) sequences were determined from 46 Proteus strains, including 388 ITS from 22 P. mirabilis strains, 211 ITS from 12 P. vulgaris strains, 169 ITS from 10 P. penneri strains, and 45 ITS from 2 P. myxofaciens strains. The Proteus strains carry mainly two types of ITS, ITS Glu (containing tRNA Glu (UUC) gene) and ITS Ile + Ala (containing tRNA Ile (GAU) and tRNA Ala (UGC) gene), and are in the forms of 28 variants with 25 genomic origins. The ITS sequences are a mosaic-like structure consisting of three conservative regions and two variable regions. The nucleotide identity of ITS subtypes in strains of the same species ranges from 96.2% to 100%. The divergence of Proteus ITS divergence was most likely due to intraspecies recombinations or horizontal transfers of sequence blocks. The phylogenetic relationship deduced from the second variable region of ITS sequences of the three facultative human pathogenic species P. mirabilis, P. vulgaris and P. penneri is similar with that based on 16S rDNA sequences, but has higher resolution to differentiate closely related P. vulgaris and P. penneri. This study is the first comprehensive study of ITS in four Proteus species and laid solid foundation for the development of high-throughput technology for quick and accurate identification of the important foodborne and nosocomial pathogens.

Structure and serological studies of the O-polysaccharide of Proteus penneri 75 : Epitopes and subgroups of Proteus serogroup O73

The O-specific polysaccharide of the lipopolysaccharide of Proteus penneri strain 75 consists of tetrasaccharide-ribitol phosphate repeating units and resembles ribitol teichoic acids of Gram-positive bacteria. The following structure of the polysaccharide was elucidated by chemical methods and 1H and 13C NMR spectroscopy: ▪ where Rib-ol is ribitol. Serological studies with polyclonal antisera showed that the same structure of the O-polysaccharide occurred in two strains: P. penneri 75 and 128. A similar structure has been established earlier for the O-polysaccharide of P. penneri 103 [Drzewiecka, D., et al., Carbohydr. Res. 337 (2002) 1535–1540]. On the basis of complex serological investigations with use of two polyclonal P. penneri 75 and 103 O-antisera, five strains could be classified into Proteus O73 serogroup: P. penneri 48, 75, 90, 103 and 128, two of which ( P. penneri 75 and 128) should be subdivided into subgroup 73a, 73b and three others ( P. penneri 48, 90 and 103) into subgroup 73a, 73c. Epitopes responsible for the cross-reactivity of P. penneri O73 strains and a related strain of P. mirabilis O20 were tentatively defined.

Structure of the O-polysaccharide of Proteus penneri 28 and Proteus vulgaris O31 and classification of P. penneri 26 and 28 in Proteus serogroup O31

The lipopolysaccharides (LPS) of Proteus penneri 28 and Proteus vulgaris O31 (PrK 55/57) were degraded with dilute acetic acid and structurally identical high-molecular-mass O-polysaccharides were isolated by gel-permeation chromatography. Sugar analysis and nuclear magnetic resonance (NMR) spectroscopic studies showed that both polysaccharides contain D-GlcNAc, 2-acetamido-2,6-dideoxy- L-glucose ( L-2-acetamido-2,6-dideoxyglucose ( N-acetylquinovosamine)) and 2-acetamido-3- O-[( S)-1-carboxyethyl]-2-deoxy- D-glucose ( N-acetylisomuramic acid) and have the following structure: where ( S)-1-carboxyethyl [a residue of ( S)-lactic acid] ( S-Lac) is an ether-linked residue of ( S)-lactic acid. The O-polysaccharide studied is structurally similar to that of P. penneri 26, which differs only in the absence of S-Lac from the GlcNAc residue. Based on the O-polysaccharide structures and serological data of the LPS, it was suggested classifying these strains in one Proteus serogroup, O31, as two subgroups: O(31a), 31b for P. penneri 28 and P. vulgaris PrK 55/57 and O31a for P. penneri 26. A serological relatedness of the LPS of Proteus O(31a), 31b and P. penneri 62 was revealed and substantiated by sharing epitope O31b, which is associated with N-acetylisomuramic acid. It was suggested that a cross-reactivity of P. penneri 28 O-antiserum with the LPS of several other P. penneri strains is due to a common epitope(s) on the LPS core.

Natural Antibiotic Susceptibility of Proteus spp., with Special Reference to P. mirabilis and P. penneri Strains

The natural susceptibility of 102 Proteus mirabilis and 35 Proteus penneri strains to 71 antibiotics was examined. Minimum inhibitory concentrations (MICs) were determined by applying a microdilution procedure in IsoSensitest broth (for all strains) and cation-adjusted Mueller Hinton broth (for some strains). P. mirabilis and P. penneri were naturally resistant to penicillin G, oxacillin, all tested macrolides, lincosamides, streptogramins, glycopeptides, rifampicin and fusidic acid. Both species were uniformly, naturally sensitive to all tested aminoglycosides, acylureidopenicillins, some cephalosporins, carbapenems, aztreonam, quinolones, sulfamethoxazole and co-trimoxazole. Species-specific differences in natural susceptibility affecting clinical assessment criteria were seen with tetracyclines, several β1-lactams, chloramphenicol and nitrufurantoin. P. mirabilis was naturally resistant to all tested tetracyclines, and was naturally sensitive to all β1-lactams, except penicillin G and oxacillin. Strains of P. penneri were naturally sensitive or of intermediate susceptibility to tetracyclines, and naturally resistant to amoxicillin (but sensitive or of intermediate susceptibility to aminopenicillins in the presence of β1-lactamase inhibitors) and some cephalosporins (i.e. cefaclor, cefazoline, loracarbef, cefurox-ime, cefotiam, and cefdinir). P. penneri was less susceptible than P. mirabilis to chloramphenicol; P. mirabilis was less susceptible than P. penneri to nitrofuran-toin. Major medium-dependent influences on the MICs were seen with fosfomycin.The present study describes a database concerning the natural antibiotic susceptibility of P. mirabilis and P. penneri strains to a range of antibiotics, which can be applied to validate forthcoming antibiotic susceptibility tests of these bacteria. It was shown that ten of fifteen amoxicillin-sensitive P. mirabilis strains produced β1-lactamases at a low level, supporting the thesis of the presence of a naturally-occurring β1-lactamase in this species. Natural susceptibility patterns are compared with those of a recent study, dealing with natural susceptibilities of species of the P. vulgaris complex.

Structure of the O-specific polysaccharide of Proteus penneri 103 containing ribitol and 2-aminoethanol phosphates

The O-specific polysaccharide of the lipopolysaccharide of Proteus penneri strain 103 was studied using 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, H-detected 1H, 13C HMQC, 1H, 31P HMQC, and HMBC experiments. It was found that the polysaccharide is built up of oligosaccharide-ribitol phosphate repeating units and thus resembles ribitol teichoic acids of Gram-positive bacteria. The following structure of the polysaccharide was established: where Etn and Rib-ol are ethanolamine and ribitol, respectively. This structure is unique among the known structures of Proteus O-antigens and, therefore, we propose classification of the strain studied into a new Proteus serogroup, O73. The molecular basis for cross-reactivity between O-antiserum against P. penneri 103 and O-antigens of P. mirabilis O33 and D52 is discussed.

The structure of the carbohydrate backbone of the rough type lipopolysaccharides from Proteus penneri strains 12, 13, 37 and 44

The following structure of the lipid A–core backbone of the rough type lipopolysaccharides (LPS) from Proteus penneri strains 12, 13, 37, and 44 was determined using NMR and mass spectroscopy and chemical analysis of the oligosaccharides obtained by mild-acid hydrolysis, alkaline O, N-deacylation, O-deacylation with hydrazine, and deamination of the LPSs: where K=H, R= PEtN, R 1=α-Hep-(1→2)-α-DDHep, and R 2=α-GalN (strains 12 and 13) or β-GlcNAc-(1→4)-α-GlcN (strains 37 and 44). LPS from each strain contained several structural variants. LPS from strain 12 contained a variant with R 1=α-DDHep, whereas LPS from strains 13, 37, and 44 contained structures with K=amide of β-GalA with putrescine or spermidine. The phosphate group at O-1 of the α-GlcN residue in the lipid part was partially substituted with Ara4N.

Structure of the core part of the lipopolysaccharides from Proteus penneri strains 7, 8, 14, 15, and 21

The core–lipid A region of the lipopolysaccharides from Proteus penneri strains 7, 8, 14, 15, and 21 was studied using NMR spectroscopy, ESI MS, and chemical analysis after alkaline deacylation, deamination, and mild-acid hydrolysis of the lipopolysaccharides. The following general structure of the major core oligosaccharides is proposed: where all sugars are in the pyranose form and have the d configuration unless otherwise stated, Hep and ddHep= l- glycero- and d- glycero- d- manno-heptose, respectively, K=H, and Q=H in strain 8 or α-Glc in strains 7, 14, 15, and 21. In addition, several minor structural variants are present, including those lacking Ara4N in strains 7 and 15 and having the α-GlcN residue N-acylated to a various degree with glycine in strains 7, 8, 14, and 21. In strain 14, there are also core oligosaccharides with K=amide of β- d-Gal pA with putrescine, spermidine, or 4-azaheptane-1,7-diamine; remarkably, these structural variants lack either the PEtN group or the α-Hep-(1→2)-α- ddHep disaccharide fragment at α- d-Gal pA. While structural features of the inner core part are shared by Proteus strains studied earlier, the outermost Q-(1→4)-α-GalNAc-(1→2)-α- ddHep-(1→6)-α-GlcN oligosaccharide unit has not been hitherto reported.

Structural and serological relatedness of the O-antigens of Proteus penneri 1 and 4 from a novel Proteus serogroup O72.

O-specific polysaccharides (O-antigens) of the lipopolysaccharides (LPS) of Proteus penneri strains 1 and 4 were studied using sugar analysis, (1)H and (13)C NMR spectroscopy, including 2D COSY, H-detected (1)H,(13)C HMQC, and rotating-frame NOE spectroscopy (ROESY). The following structures of the tetrasaccharide (strain 1) and pentasaccharide (strain 4) repeating units of the polysaccharides were established: [reaction: see text]. In the polysaccharide of P. penneri strain 4, glycosylation with the lateral Glc residue (75%) and O-acetylation of the lateral GalNAc residue (55%) are nonstoichiometric. This polysaccharide contains also other, minor O-acetyl groups, whose positions were not determined. The structural similarity of the O-specific polysaccharides was consistent with the close serological relatedness of the LPS, which was demonstrated by immunochemical studies with O-antisera against P. penneri 1 and 4. Based on these data, it was proposed to classify P. penneri strains 1 and 4 into a new Proteus serogroup, O72, as two subgroups, O72a and O72a,b, respectively. Serological cross-reactivity of P. penneri 1 O-antiserum with the LPS of P. penneri 40 and 41 was substantiated by the presence of an epitope(s) on the LPS core region shared by all P. penneri strains studied.

New structures of the O-specific polysaccharides of bacteria of the genus Proteus. 1. Phosphate-containing polysaccharides.

The O-specific polysaccharide chains (O-antigens) of the lipopolysaccharides of five Proteus strains, P. vulgaris O17, P. mirabilis O16 and O33, and P. penneri 31and 103, were found to contain phosphate groups that link the non sugar components, e.g., ethanolamine and ribitol. The polysaccharides of P. mirabilis O16 and P. penneri 103 include ribitol phosphate in the main chain and thus resemble ribitol teichoic acids of Gram-positive bacteria. The structures of the polysaccharides were elucidated using NMR spectroscopy, including two-dimensional 1H,1H correlation spectroscopy (COSY and TOCSY), nuclear Overhauser effect spectroscopy (NOESY or ROESY), and H-detected 1H,13C and 1H,31P heteronuclear multiple-quantum coherence spectroscopy (HMQC), along with chemical methods. The structures determined are unique among the bacterial polysaccharides and, together with the data obtained earlier, represent the chemical basic for classification of Proteus strains. Based on structural similarities of the O-specific polysaccharides and serological relationships between the O-antigens, we propose to extend Proteus serogroups O17 and O19 by including P. penneri strains 16 and 31,respectively.

The structure of the carbohydrate backbone of the core–lipid A region of the lipopolysaccharide from Proteus penneri strain 40: new Proteus strains containing open-chain acetal-linked N-acetylgalactosamine in the core part of the LPS

Analysis of the core part of the LPS from several strains of Proteus revealed that P. penneri strains 2, 11, 19, 107, and P. vulgaris serotypes O4 and O8 have the same structure with a new type of linkage between monosaccharides–an open-chain acetal — that was previously determined for P. vulgaris OX2 and P. penneri 17. The LPS from P. penneri strain 40 contains the same structure substituted with one additional monosaccharide: where (1 S)-Gal aNAc 1 The abbreviation Gal oNAc was used in two previous reports, 4,5 but the alternative designation Gal aNAc ( a denoting acyclic, acetal-linked, or aldehydo) is recommended for future use. Editor. 1 is a residue of N-acetyl- d-galactosamine in the open-chain form. It is connected as a cyclic acetal to positions 4 and 6 of the galactosamine residue having a free amino group. All other sugars are in the pyranose form.

The structure of the core part of Proteuspenneri strain 16 lipopolysaccharide

The structure of the carbohydrate backbone of the lipid A-core region of the lipopolysaccharide (LPS) from Proteus penneri strain 16 was determined using NMR and chemical analysis of the core oligosaccharide, obtained by mild acid hydrolysis of the LPS, and of the products of alkaline deacylation of the LPS: Incomplete substitution is indicated by bold italics. All sugars are in the pyranose form, α-Hep is the residue of l- glycero-α- d- manno-Hep, α- dd-Hep is the residue of d- glycero-α- d- manno-Hep, Bu is the ( R)-3-hydroxybutyryl residue.

Use of randomly amplified polymorphic DNA (RAPD) analysis for identification of Proteus penneri.

The RAPD assay applied in our study confirmed the taxonomic position of P. penneri bacteria. The low annaealing temperature and the 22- oligonucleoide primers used in our study resulted in PCR patterns highly specific for P. penneri species. The analysis of a number of Proteus clinical isolates as well as other Gram-negative bacteria showed that P. penneri strains composed a clonally related group which can be distinguished from other bacteria including P. mirabilis and P. vulgaris. Moreover, the RAPD assay performed by us could be useful in one step PCR based identification of P. penneri strains.

Determination of genetic diversity of Proteus penneri strains using rep-PCR.

The results of our study have shown that REP-like repetitive DNA elements are present in the genome of Proteus strains and the rep-PCR technique is useful for generating DNA fingerprints of Proteus penneri strains. Beside evident differences in DNA band patterns ofthe strains there were bands specific for all of the Proteus penneri isolates. Analysis of these results showed a large degree ofgenetic heterogenity among these bacteria. Furthermore, a PCR fingerprint technique was used in this study in order to characterise clinical Proteus penneri strains and to determine the applicability of this technique for identification and comparison of Proteus strains from clinical and other biological materials, for diagnostic and epidemiological purposes.

Structure of a 2-aminoethyl phosphate-containing O-specific polysaccharide of Proteus penneri 63 from a new serogroup O68.

Lipopolysaccharide of Proteus penneri strain 63 was degraded by mild acid to give a high molecular mass O-specific polysaccharide that was isolated by gel-permeation chromatography. Sugar and methylation analyses and NMR spectroscopic studies, including two-dimensional 1H, 1H COSY, TOCSY rotating-frame NOE spectroscopy, H-detected 1H,13C and 1H,31P heteronuclear multiple-quantum coherence (HMQC), and 1H, 13C HMQC-TOCSY experiments, demonstrated the following structure of the polysaccharide: where FucNAc is 2-acetamido-2,6-dideoxygalactose and PEtn is 2-aminoethyl phosphate. The polysaccharide studied shares some structural features, such as the presence of D-GlcNAc6PEtn and an alpha-L-FucNAc-(1-->3)-D-GlcNAc disaccharide, with other Proteus O-specific polysaccharides. A marked cross-reactivity of P. penneri 63 O-antiserum with P. vulgaris O12 was observed and substantiated by a structural similarity of the O-specific polysaccharides of the two strains. In spite of this, the polysaccharide of P. penneri 63 has the unique structure among Proteus O-antigens, and therefore a new, separate serogroup, O68, is proposed for this strain.

New serogroups of the genus Proteus consisting of Proteus penneri strains only. Determination of some LPS epitopes responsible for specificity.

On the basis of serological investigations of 133 Proteus strains and structural analysis of P.penneri O-polysaccharides the following new Proteus serogroups have been proposed (Table 6.).