Extended-spectrum beta-lactamase enzymes in clinical isolates of Enterobacter species from Lagos, Nigeria.

Over a 9-month period, 8 of 40 nonduplicate isolates of Enterobacter spp. producing extended-spectrum beta-lactamase (ESBL) were detected for the first time from two hospitals in Lagos, Nigeria. Microbiologic and molecular analysis confirmed the presence of ESBL. Only four isolates transferred ESBL resistance as determined by the conjugation test, and pulsed-field gel electrophoresis showed genetically unrelated isolates.

Escherichia coli survival in groundwater and effluent measured using a combination of propidium iodide and the green fluorescent protein.

AIMS: The aim of this study was to deterimine the survival of an enteric bacterium in anaerobic groundwater and effluent microcosms using the green fluorescent protein (GFP) marker gene in combination with the viability indicator propidium iodide (PI). METHODS AND RESULTS: The pEGFP vector (Clontech) was transformed into Escherichia coli DH5alpha and was stable for at least 100 generations of growth in nonselective medium at 28 degrees C and 37 degrees C. Using an epifluorescent microscope, GFP cells could be detected under blue light (450-490 nm) and the numbers of PI-positive GFPs could be detected under green light (530-560 nm). GFP-tagged E. coli could be detected for at least 132 d in sterilized water microcosms. GFP fluorescence was not lost from the culturable cell population for the duration of the experiment. However, a slow decline in the number of GFP-fluorescent cells in sterilized groundwater was observed. Escherichia coli die-off and loss of green fluorescence was more rapid in nonsterilized waters than in sterilized. Viable numbers of the GFP-tagged E. coli determined by PI counterstaining were compatible with numbers of colony-forming units. CONCLUSIONS: The long-term survival of E. coli and maintainance of GFP-conferred fluorescence in these cells was demonstrated in both groundwater and effluent, under sterilized conditions. However, severe starvation and/or the presence of indigenous microorganisms were found to be factors affecting the maintenance of fluorescence in dead or dying cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the successful application of PI with GFP-tagging to monitor long-term bacterial survival in nutrient-limited conditions and mixed microbial populations.

The detection and recovery of Erysipelothrix spp. in meat and abattoir samples in Western Australia.

AIMS: To investigate the occurrence of Erysipelothrix rhusiopathiae and other Erysipelothrix spp. in abattoir and meat samples in Western Australia. METHODS AND RESULTS: Samples were collected from various parts of pig and sheep carcasses, as well as different sections of slaughtering line, pen soil and effluent. Previously evaluated culture methods were applied for the isolation of Erysipelothrix spp., in conjunction with phenotypic and genotypic detection and identification procedures. Of 109 samples from the two abattoirs, 35 (32.1%) were Erysipelothrix genus-specific PCR-positive. These came from swabs of animal exterior surfaces and joints, slaughtering areas, pig pen soil and abattoir effluent. Four samples (3.7%) from sheep arthritic joints and pig abattoir effluent were also E. rhusiopathiae species-specific PCR-positive. Of 123 carcass washing samples, 12 (9.8%) were genus-specific PCR-positive, and these came from all five kinds of meat samples tested, including beef, lamb, mutton, pork and chicken. Four of them (3.3%) were also species-specific PCR-positive. A total of 25 isolates was recovered from the samples, of which seven were identified as E. rhusiopathiae, seven were consistent with E. tonsillarum, and the remaining 11 were other species of Erysipelothrix. CONCLUSIONS: Erysipelothrix spp. can still be isolated and identified from specimens of animal origin with relative ease, provided that appropriate cultural and molecular procedures are used. Clinical microbiology laboratories may need to improve their diagnostic protocols. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms that E. rhusiopathiae and other species of Erysipelothrix continue to colonize and contaminate farmed animals and animal products. Erysipelothrix infection still poses a potential threat to the economy of the farmed animal industry, as well as being a potential human public health hazard.

Desulfosporosinus meridiei sp. nov., a spore-forming sulfate-reducing bacterium isolated from gasolene-contaminated groundwater.

Eight strains of spore-forming, sulfate-reducing bacteria, isolated from groundwater contaminated with motor fuel [mostly benzene, toluene ethylbenzene and xylene (BTEX) compounds] in sandy soil near Perth, Australia, were closely related to Desulfosporosinus (previously Desulfotomaculum) orientis DSM 765T (95.3-97.3% 16S rDNA sequence similarity). Whole-cell fatty acids were dominated by even-carbon, straight-chain saturated and mono-unsaturated fatty acids, in particular 16:0, 16:1cis9, 14:0 and 18:1cis11. The strains grew at temperatures between 4 and 42 degrees C and in medium containing up to 4% NaCl. The eight strains clustered into two main groups based on phylogeny, randomly amplified polymorphic DNA (RAPD)-PCR patterns and nutritional characteristics. Representatives of the two groups, strain S5 (group A) and strain S10T (group B) had 81% DNA-DNA homology with each other and therefore should be accommodated in the same species. Strain S10T had less than 38% homology with Desulfosporosinus orientis DSM 765T, the most closely phylogenetically related type strain available. The new strains were distinguished from Desulfosporosinus orientis DSM 765T by different banding patterns in a RAPD-PCR, and phenotypically by their inability to utilize fumarate as a carbon and energy source with sulfate as the electron acceptor and by their lower tolerance to NaCl. The DNA G+C contents were 46.8 and 46.9 mol% for strains S5 and S10T, respectively (Desulfosporosinus orientis DSM 765T 45.9 mol%). It is proposed that these new strains be placed in a new species of the genus Desulfosporosinus. The name Desulfosporosinus meridiei is proposed, with strain S10T as the type strain (= DSM 13257T = NCIMB 13706T).

Identification of genes associated with copper tolerance in an adhesion-defective mutant of Aeromonas veronii biovar sobria.

TnphoA mutagenesis was used to identify adhesins of Aeromonas veronii biovar sobria 3767, a strain isolated from a diarrhoeal stool specimen. Six mutants, from a library of 154, exhibited significantly reduced levels of adhesion to HEp-2 cells. Primers to the terminal regions of TnphoA were used for inverse PCR and the product from one mutant was cloned into pBluescript and partial sequence data obtained. Scanning GenBank and EMBL data bases revealed DNA sequence similarity to the copA gene of Pseudomonas syringae pv. tomato which confers resistance to copper and other heavy metals. The transposon was located within the copA gene and the mutant exhibited a reduced tolerance to copper. Primer walking, using the inverse PCR product as a template, revealed three open reading frames (ORFs) copA, B and C in A. veronii biovar sobria 3767. The predicted amino acid sequences of ORFs A and B had significant homology (55 and 34% respectively) to the copA and B proteins of P. syringae. No amino acid or DNA sequence homology existed between ORF C of strain 3767 and any other gene in the data bases scanned. Further analysis of the nucleotide sequence failed to reveal the presence of typical copper regulatory genes within the vicinity of the Aeromonas sequence. The association between copper tolerance and adhesion in A. veronii biovar sobria requires further study.

Spore-forming, Desulfosporosinus-like sulphate-reducing bacteria from a shallow aquifer contaminated with gasoline.

Previous studies on the geochemistry of a shallow unconfined aquifer contaminated with hydrocarbons suggested that the degradation of some hydrocarbons was linked to bacterial sulphate reduction. There was attenuation of naphthalene, 1,3,5-trimethylbenzene (TMB), toluene, p-xylene and ethylbenzene in the groundwater with concomitant loss of sulphate. Here, the recovery of eight strains of sulphate-reducing bacteria (SRB) from the contaminated site is reported. All were straight or curved rod-shaped cells which formed endospores. Amplification and sequencing of the 16S rDNA indicated that the strains were all sulphate reducers of the Gram-positive line of descent, and were most closely related to Desulfosporosinus (previously Desulfotomaculum) orientis DSM 8344 (97-98.9% sequence similarity). The strains clustered in three phylogenetic groups based on 16S rRNA sequences. Whole cell fatty acid compositions were similar to those of D. orientis DSM 8344, and were consistent with previous studies of fatty acids in soil and groundwater from the site. Microcosms containing groundwater from this aquifer indicated a role for sulphate reduction in the degradation of [ring-UL-14C]toluene, but not for the degradation of [UL-14C]benzene which could also be degraded by the microcosms. Adding one of the strains that was isolated from the groundwater (strain T2) to sulphate-enriched microcosms increased the rate of toluene degradation four- to 10-fold but had no effect on the rate of benzene degradation. The addition of molybdate, an inhibitor of sulphate reduction, to the groundwater samples decreased the rate of toluene mineralization. There was no evidence to support the mineralization of [UL-14C]benzene, [ring-UL-14C]toluene or unlabelled m-xylene, p-xylene, ethylbenzene, TMB or naphthalene by any of the strains in pure culture. Growth of all the strains was completely inhibited by 100 micromol l-1 TMB.

Moraxella catarrhalis: clinical significance, antimicrobial susceptibility and BRO beta-lactamases.

Moraxella catarrhalis is an important pathogen of humans. It is a common cause of respiratory infections, particularly otitis media in children and lower respiratory tract infections in the elderly. Colonisation of the upper respiratory tract appears to be associated with infection in many cases, although this association is not well understood. Nosocomial transmission is being increasingly documented and the emergence of this organism as a cause of bacteremia is of concern. The widespread production of a beta-lactamase enzyme renders Moraxella catarrhalis resistant to the penicillins. Cephalosporins and beta-lactamase inhibitor combinations are effective for treatment of beta-lactamase producers, and the organism remains nearly universally susceptible to the macrolides, fluoroquinolones, tetracyclines and the combination of trimethoprim and sulfamethoxazole. Two major beta-lactamase forms, BRO-1 and BRO-2, have been described on the basis of their isoelectric focusing patterns. The BRO-1 enzyme is found in the majority of beta-lactamase-producing isolates and confers a higher level of resistance to strains than BRO-2. The BRO enzymes are membrane associated and their production appears to be mediated by chromosomal determinants which are transmissible by an unknown mechanism. The origin of these novel proteins is unknown.

Characterization of the roles of hemolysin and other toxins in enteropathy caused by alpha-hemolytic Escherichia coli linked to human diarrhea.

Escherichia coli strains producing alpha-hemolysin have been associated with diarrhea in several studies, but it has not been clearly demonstrated that these strains are enteropathogens or that alpha-hemolysin is an enteric virulence factor. Such strains are generally regarded as avirulent commensals. We examined a collection of diarrhea-associated hemolytic E. coli (DHEC) strains for virulence factors. No strain produced classic enterotoxins, but they all produced an alpha-hemolysin that was indistinguishable from that of uropathogenic E. coli strains. DHEC strains also produced other toxins including cytotoxic necrotizing factor 1 (CNF1) and novel toxins, including a cell-detaching cytotoxin and a toxin that causes HeLa cell elongation. DHEC strains were enteropathogenic in the RITARD (reversible intestinal tie adult rabbit diarrhea) model of diarrhea, causing characteristic enteropathies, including inflammation, necrosis, and colonic cell hyperplasia in both small and large intestines. Alpha-hemolysin appeared to be a major virulence factor in this model since it conferred virulence to nonpathogenic E. coli strains. Other virulence factors also appear to be contributing to virulence. These findings support the epidemiologic link to diarrhea and suggest that further research into the role of DHEC and alpha-hemolysin in enteric disease is warranted.

The identification of response regulators of Branhamella catarrhalis using PCR.

Potential response regulator gene fragments from the genome of Branhamella (Moraxella) catarrhalis were isolated by PCR using degenerate oligonucleotide primers. DNA sequence analysis of several cloned PCR products with similar restriction endonuclease analysis (REA) patterns revealed that the cloned gene fragment had significant homology to members of the OMPR sub-family of response regulator genes, including 61% identity with the phoB gene of Haemophilus influenzae. The derived amino acid sequence showed greatest similarity to the PhoB response regulator protein of Pseudomonas aeruginosa. Characterisation of this phoB homologue and of other response regulators identified in this study should provide new knowledge of the physiology and pathogenic mechanisms of B. catarrhalis.

Identification of clinical isolates of indole-positive Klebsiella spp., including Klebsiella planticola, and a genetic and molecular analysis of their beta-lactamases.

In a collection of 43 indole-positive Klebsiella clinical isolates, which were initially identified as Klebsiella oxytoca, there were 18 isolates which exhibited a pattern characteristic of extended-spectrum beta-lactamase (ESBL) resistance. This study aimed to confirm their identity by biochemical tests and by PCR and to determine the genetic basis for their resistance to the beta-lactams and broad-spectrum cephalosporins. Chromosomal beta-lactamase genes were analyzed by PCR, and plasmid-mediated beta-lactamase genes were analyzed by conjugation and transformation. There were 39 isolates which grew on melezitose but failed to grow on 3-hydroxybutyrate, confirming them as K. oxytoca. PCR analysis of their beta-lactamase genes divided these isolates into two groups, the bla(OXY-1) group and the bla(OXY-2) group. Each group had beta-lactamases with different isoelectric points; the bla(OXY-1) group had beta-lactamases with isoelectric points at 7.2, 7.8, 8.2, and 8.8, and the more common bla(OXY-2) group had beta-lactamases with pIs at 5.2, 5.4 (TEM-1), 5.7, 5.9, 6.4, and 6.8. A pI of 5.2 was the most frequently detected and accounted for 59% of all the bla(OXY-2) beta-lactamases. Hyperproduction of clavulanate-inhibited chromosomal beta-lactamases was detected in 17 K. oxytoca isolates, resulting in an ESBL phenotype. K. oxytoca isolates having a plasmid-mediated genetic basis for their ESBL phenotype were not found, confirming that, in K. oxytoca, plasmids are rarely involved in conferring resistance to the newer cephalosporins. Four isolates proved to be isolates of K. planticola in which the beta-lactamase genes failed to react with the primers used in the PCR. One K. planticola isolate contained a transferable plasmid harboring the SHV-5 beta-lactamase gene and showed an ESBL phenotype, while the other non-ESBL K. planticola isolates contained chromosomal beta-lactamases with isoelectric points at 7.2, 7.7, and 7.9 plus 7.2.

Staphylococcus aureus but not Staphylococcus epidermidis can acquire iron from transferrin.

Staphylococci grow and cause infection under the iron-restricted conditions found in vivo. They therefore must possess mechanisms to obtain iron for metabolism from this environment. To determine if staphylococci can extract iron bound to human transferrin, we labelled transferrin with 55Fe and performed uptake assays on cells grown in iron-restricted and iron-plentiful conditions. Growing cultures of Staphylococcus aureus NCTC 8532 could take up radioactive iron during mid- to late-exponential phase of growth. This process was iron-regulated and did not require direct contact between the cell and the labelled transferrin. Siderophore production was detected during this phase, but reductase or protease activity was not. S. epidermidis ATCC 14990 could not access 55Fe bound to transferrin, nor did this isolate produce siderophore, reductase or protease. This difference in the ability to acquire iron bound to transferrin may contribute to the increased virulence of S. aureus when compared to S. epidermidis.

Production of siderophore by coagulase-negative staphylococci and its relation to virulence.

The ability to produce siderophore is considered to be a virulence factor for many pathogenic bacteria. To determine if siderophore production by coagulase-negative staphylococci (CNS) was related to virulence, 40 clinical isolates of CNS cultured from peritoneal dialysis fluid were compared with 38 commensal skin isolates. Siderophore activity was detected using the chrome azurol S liquid assay. Using precursor studies, Staphylococcus epidermidis isolates were shown to be more likely to produce the siderophore staphyloferrin A. Production of staphyloferrin B amongst non-Staphylococcus epidermidis species was associated with clinical isolates rather than commensal isolates, and therefore may play a role in pathogenicity.

Structural comparison and epitope analysis of outer-membrane protein PIA from strains of Neisseria gonorrhoeae with differing serovar specificities.

The sequences of the por genes, encoding outer-membrane protein PI, have been obtained from a number of strains of Neisseria gonorrhoeae that express PIA molecules with differing serovar specificities. The inferred amino acid sequences of the mature proteins each comprise 308 residues and show considerable homology, with the degree of sequence variation between PIA molecules being considerably less than seen previously with PIB, but more evenly distributed throughout the molecule. The positions of sequence variation are largely confined to the regions predicted to form one of eight surface-exposed loops, suggesting a more widespread distribution of potential antigenic diversity. The deduced amino acid sequences were used to synthesize peptides for epitope mapping experiments. Some epitopes responsible for serovar specificity or recognized by bactericidal monoclonal antibodies could be identified on the basis of their reactivity with simple linear peptides, whilst others recognized conformational epitopes. By comparison of sequence differences with mAb reactivity it was possible to identify regions that appear to contribute to such determinants, including separated regions of the molecule which together were required for the formation of the conformational epitopes. All the epitopes identified lie at or close to the apices of the predicted surface-exposed loops 1, 3, 6, or 8, focusing attention on these regions as accessible targets for immune attack.

Purification and characterization of the urease enzymes of Helicobacter species from humans and animals.

The urease enzymes of Helicobacter pylori, H. mustelae, H. felis, and H. nemestrinae have been purified to homogeneity by affinity chromatography and characterized. The native urease enzymes of the four organisms were found to be almost identical, with a pI of 6.1 and molecular masses of 480 to 500 kDa, as determined by electrophoretic mobility in nondenaturing polyacrylamide gels. Transmission electron microscopy of the native urease showed it to be a molecule approximately 13 nm in diameter, with hexagonal symmetry. Denaturation studies indicated that each urease enzyme molecule was composed of two nonidentical subunits with molecular masses of approximately 64 and 30 kDa. The subunits were present in a 1:1 ratio, suggesting a hexameric stoichiometry for the native molecule. The predicted molecular mass of H. pylori urease, based on subunit molecular weight and stoichiometry, is 568 kDa. N-terminal amino acid sequencing of the enzyme subunits from the four species revealed high levels of homology. The large subunits (UreB) were found to be 92 to 100% homologous, and the small subunits (UreA) were 75 to 95% homologous over the first 12 to 20 residues. The high degree of homology suggests a common ancestral origin and an important role for the urease enzymes of these organisms.

Characterisation of the urease from Helicobacter pylori and comparison with the ureases from related spiral gastric bacteria.

The urease enzyme of Helicobacter pylori was partially purified from whole cell extracts and found to have a molecular weight of 484 +/- 12 kDa. Ten monoclonal antibodies (mAbs) were produced against four different epitopes of the native enzyme. These mAbs also recognised the ureases of H. pylori-like organisms isolated from monkeys and pigs and the H. mustelae urease from ferrets. The urease enzymes of each of these organisms were found to be of the same molecular weight. The urease enzyme of H. pylori consisted of two subunits of 68.2 and 31.3 kDa.

Soluble and cell-associated haemagglutinins of Helicobacter (Campylobacter) pylori.

Some plate-grown strains of Helicobacter (Campylobacter) pylori that were harvested into phosphate-buffered saline and left for 1 h released soluble haemagglutinins. These caused high-titre agglutination of human and guinea-pig erythrocytes, whereas chicken, sheep and bovine erythrocytes were agglutinated at various titres. Six of 10 strains which had been subcultured repeatedly did not possess soluble haemagglutinins. Slide agglutination of bacterial suspensions demarcated the strains into two groups; Group 1 gave strong agglutination with most types of erythrocyte, Group 2 did not. By microtitration assay, all Group-1 strains but only two Group-2 strains produced a soluble haemagglutinin. Cell-associated haemagglutinins were found by microtitration assay in all strains of H. pylori, but higher titres were found within Group-1 strains. The supernates of broth-grown, shaken cultures also showed the presence of soluble haemagglutinins, with higher titres for recently isolated strains. Pre-treatment of human erythrocytes with neuraminidase from Arthrobacter ureafaciens and Clostridium perfringens abolished haemagglutination by the soluble, but not by the cell-associated haemagglutinin. The soluble haemagglutinin was inhibited by sialoproteins containing predominantly the N-acetylneuraminyl (2-3) galactopyranosyl [NeuAc(2-3)Gal] structure, fetuin, glycophorin and bovine N-acetylneuraminyl-lactose (NeuAc-Lac). Transferrin and human NeuAc-Lac, which contain predominantly the N-acetylneuraminyl (2-6) galactopyranosyl [NeuAc(2-6)Gal] structure were not inhibitory. However, bovine submaxillary mucin (BSM) was strongly inhibitory; it contains several structures with sialic acid linked 2-6 to oligosaccharides. These results suggest that the soluble haemagglutinin recognises a NeuAc(2-3)Gal structure, but has high affinity for another, as yet undetermined, sialic acid-containing structure.

Distribution of plasmid maintenance regions among IncFIV plasmids.

The distribution of the IncFI basic replicons among IncFIV plasmids was assessed by DNA hybridization. In addition these and 20 other plasmids from 16 incompatibility groups were screened for the presence of IncIV, an incompatibility determinant recently found on the IncFIV plasmid R124. The IncIV determinant was found commonly but not universally among the IncFIV plasmids. It was also detected on the IncFI reference plasmid R386 and plasmids from IncB, IncI alpha and IncI gamma. The frequency and distribution of IncFI replicons among the IncFIV plasmids is similar to that observed in other F groups. The similarity of the IncFIV plasmids to plasmids of the other IncF groups and the failure to find replicons unique to IncFIV plasmids indicates that their division into a separate incompatibility group is not justified.

The epidemiology of Neisseria gonorrhoeae isolates from Western Australia based on antibiotic resistance and plasmid profiles.

Two collections of 90 distinct isolates of Neisseria gonorrhoeae were examined with respect to their plasmid profiles, auxotypes and antibiotic sensitivities to penicillin, erythromycin, streptomycin, spectinomycin, sulphamethoxazole, tetracycline and trimethoprim. The two series, collected from 1976-1981 and from 1985-1986 possessed similar sensitivity patterns except for erythromycin and trimethoprim. The earlier collection of penicillinase producing N. gonorrhoeae (PPNG) was found to be significantly more resistant to both these antibiotics. Within each series the PPNG isolates were more resistant to penicillin and streptomycin than the non-PPNG. All PPNG isolates had the 4.4 X 10(6) dalton penicillinase plasmid and frequently exhibited an auxotype which included a requirement for proline. The frequency of the 24.5 X 10(6) dalton transfer plasmid in the non-PPNG was found to have increased threefold from the early collection to the later one while amongst the PPNG the frequency of the transfer plasmid was seen to decrease. Auxotyping results indicated that the transfer plasmid played a significant role in the spread of the penicillinase plasmid in the early collection. In the later collection a transfer-plasmid-free strain, which required only proline, comprised almost half of the penicillinase producers and this PPNG strain now appears to be adapted to our environment.