Comparison of automated ribotyping and pulsed-field gel electrophoresis for subtyping of Vibrio cholerae

Aims:  To compare the discriminatory power of an automated ribotyping method for Vibrio cholerae subtyping with the pulsed-field gel electrophoresis (PFGE), to evaluate the possibility of automated ribotyping in use of outbreak investigations and surveillance of cholera.
Methods and Results:  Eight-one epidemiologically unrelated isolates of V. cholerae , and 19 isolates from seven cholera outbreaks were used as the panels. When comparing the two methods using the epidemiologically unrelated isolates, automated ribotyping using Pvu II distinguished 38 different ribotypes with a D -value of 0·8956. When combined with serotyping, the D -value is 0·9466. However, PFGE with Not I and Sfi I digestions had higher D -values of 0·9951 and 0·9948, respectively. PFGE could cluster the isolates from each outbreak into the same pattern, and distinguish different patterns from different outbreaks, whereas automated ribotyping had lower discriminatory ability.
Conclusions:  The automated ribotyping has lower discriminatory ability compared to PFGE, and is limited to application in V. cholerae subtyping and outbreak investigation.
Significance and Impact of the Study:  The study evaluated the limitation in subtyping of automated ribotyping for V. cholerae , and raise the question of improvement for the automated ribotyping in subtyping.     

Genotyping of Campylobacter coli isolated from humans and retail meats using multilocus sequence typing and pulsed-field gel electrophoresis

Aims:  To determine the antimicrobial resistant profiles and clonality of Campylobacter coli isolated from clinically ill humans and retail meats.
Methods and Results:  A total of 98 C. coli isolates (20 from humans and 78 from retail meats) were phenotypically characterized. Antimicrobial susceptibility testing was done using agar dilution method for ciprofloxacin, gentamicin, erythromycin and doxycycline. Seventy C. coli isolates including humans ( n  = 20) and retail meats ( n  = 50) were genotyped by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Resistance to ciprofloxacin was found in 29% and 15% of isolates from retail meats and humans. We observed 61 PFGE profiles using two enzymes ( Sma I, Kpn I) with an Index of discrimination of 0·99, whereas MLST generated 37 sequence types. Two clonal complexes were identified with 58 (82%) C. coli isolates clustered in the ST-828 complex.
Conclusions:  Resistance to ciprofloxacin and erythromycin was identified in C. coli obtained from retail meats and ill humans. PFGE typing of C. coli isolates was more discriminatory than MLST. Grouping of C. coli isolates (82%) by MLST in ST-828 clonal complex indicates a common ancestry.
Significance and Impact of the Study:  A high frequency of resistance found to ciprofloxacin and erythromycin is concerning from food safety perspective. PFGE using single or double restriction enzymes was found to be more discriminatory than MLST for genotyping C. coli . Overall, the C. coli populations recovered from humans and retail meats were genotypically diverse.     

Host range and lytic capability of four bacteriophages against bovine and clinical human isolates of Shiga toxin-producing Escherichia coli O157:H7

Aims:  To evaluate host range and lytic capability of four bacteriophages (rV5, wV7, wV8 and wV11) against Escherichia coli O157:H7 (STEC O157:H7) from cattle and humans.
Methods and Results:  Four hundred and twenty-two STEC O157:H7 isolates (297 bovine; 125 human) were obtained in Alberta, Canada. The four phages were serially diluted and incubated for 5 h with overnight cultures of STEC O157:H7 to estimate their multiplicity of infection (MOI). All bovine STEC O157:H7 were subjected to pulsed-field gel electrophoresis (PFGE) and phage typing (PT). Phage wV7 lysed all human and bovine isolates irrespective of PFGE genotype or PT phenotype and exhibited the lowest MOI (0·004–0·006, P  < 0·0001) of all phages. Phages rV5 and wV11 exhibited a lower MOI (0·002–0·04, P  < 0·0001) than did phage wV8 (25–29) and they had a narrower host range than wV7 or wV8. Phages rV5, wV11 and wV8 lysed 342 (81·0%), 321 (76·1%) and 407 (96·4%), respectively, of the 422 isolates. Susceptibility of bovine STEC O157:H7 to rV5, w11 and wV8 was influenced by PFGE genotype and/or PT phenotype.
Conclusions:  Phages exhibited activity against the majority of bovine and human STEC O157:H7 isolates. PFGE genotype and/or PT phenotype of the host-target influenced their vulnerability to phage attack. Susceptibility of bovine STEC O157:H7 to phage may also differ among farms. Both lytic capability and host range should be considered in the selection of therapeutic phage for on-farm control of STEC O157:H7.
Significance and Impact of the Study:  The present work indicates that a four-phage cocktail should be equally effective at mitigating STEC O157:H7 isolates both of bovine and of human origin. Given that some STEC O157:H7 exhibited resistance to some but not all phages, a phage cocktail is the logical approach to efficacious on-farm therapy.     

Isolation of bovine intestinal Lactobacillus plantarum and Pediococcus acidilactici with inhibitory activity against Escherichia coli O157 and F5

Aims:  The growth rate of bovine lactic acid bacteria (LAB) in five different culture conditions, and their inhibitory activity against Escherichia coli O157 and F5 in two assays was assessed to identify LAB for potential prophylactic use in cattle.
Methods and Results:  106 bovine-derived faecal/intestinal LAB were tested in vitro for tolerance to pH 2·0, pH 4·0, 0·15% and 0·3% bile, aerobic incubation, and for inhibitory activity against E. coli O157 ( n  = 3) and F5 ( n  = 1). While no LAB grew at pH 2·0, LAB survivability varied between 35% and 100% on the other tests. Exactly 7·6% (8/106) of LAB supernatants inhibited the growth of E. coli in two assays, whereas 6·6% (7/106) of isolates enhanced the growth of all E. coli strains. Partial 16s rRNA gene sequencing of six best isolates (95th percentile) revealed that five were Lactobacillus plantarum and one Pediococcus acidilactici.
Conclusion:  Lactobacillus plantarum with acid/bile and aerobic resistance and inhibitory activity against E. coli O157 and F5 inhabit the intestinal tract of healthy cattle. Some LAB may enhance E. coli growth.
Significance and Impact of the Study:  Lactobacillus plantarum and P. acidilactici are natural plant micro-organisms and studied silage inoculants. Their identification from gastrointestinal samples of healthy cattle is prophylactically promising.     

Comparison of antimicrobial resistance patterns between clinical and sewage isolates in a regional hospital in Taiwan

Aims:  To compare bacterial populations and antimicrobial resistance patterns between clinical and sewage isolates from a regional hospital in northern Taiwan. The dissemination of antibiotic-resistant bacteria from hospital compartments to the hospital sewage treatment plant was examined.
Methods and Results:  A total of 1020 clinical isolates and 435 sewage isolates were collected between July and September 2005. The percentages of Gram-negative bacteria from the clinical and sewage isolates were 87·2% and 91·0%, respectively ( P  =   0·033). Escherichia coli were the leading bacterial isolates in both groups. Antimicrobial susceptibility testing showed a significant difference ( P  <   0·001) in resistance to ampicillin (85·6% vs 94·1%), ampicillin/sulbactam (31·7% vs 55·4%), cefazolin (29·2% vs 71·5%) and cefuroxime (20·7% vs 61·9%) between clinical and sewage coliform isolates, respectively.
Conclusions:  The sewage isolates had higher antimicrobial resistance rates than the clinical isolates from the same hospital.
Significance and Impact of the Study:  The low efficacy of the hospital sewage treatment may contribute to the dissemination of multidrug resistant bacteria from this hospital compartments to the environment. Practices which limit the disposal of antimicrobial agents into the wastewater system may be the possible measure to prevent the selection of multidrug-resistant bacteria from sewage treatment plants.     

Detection of metallo-β-lactamases-encoding genes in environmental isolates of Aeromonas hydrophila and Aeromonas jandaei

Aims:  To determine the prevalence and expression of metallo-β-lactamases (MBL)-encoding genes in Aeromonas species recovered from natural water reservoirs in southeastern Brazil.
Methods and Results:  Eighty - seven Aeromonas isolates belonging to Aeromonas hydrophila ( n  =   41) and Aer. jandaei ( n  =   46) species were tested for MBL production by the combined disk test using imipenem and meropenem disks as substrates and EDTA or thioglycolic acid as inhibitors. The presence of MBL genes was investigated by PCR and sequencing using new consensus primer pairs designed in this study. The cphA gene was found in 97·6% and 100% of Aer. hydrophila and Aer. jandaei isolates, respectively, whereas the acquired MBL genes bla _IMP , bla _VIM and bla _SPM-1 were not detected. On the other hand, production of MBL activity was detectable in 87·8% and 10·9% of the cphA -positive Aer. hydrophila and Aer. jandaei isolates respectively.
Conclusions:  Our results indicate that cphA seems to be intrinsic in the environmental isolates of Aer. hydrophila and Aer. jandaei in southeastern Brazil, although, based on the combined disk test, not all of them are apparently able to express the enzymatic activity.
Significance and Impact of the Study:  These data confirm the presence of MBL-producing Aeromonas species in natural water reservoirs. Risk of waterborne diseases owing to domestic and industrial uses of freshwater should be re-examined from the increase of bacterial resistance point of view.     

Application of pulsed-field gel electrophoresis for the discrimination of leptospiral isolates in Brazil

Aims:  Leptospirosis is a public health problem worldwide. Traditionally, microscopic agglutination test (MAT) and cross-agglutinin absorption test (CAAT) are used to identify leptospires. However, these techniques are laborious and time-consuming, requiring the maintenance of a collection of more than 200 reference strains and correspondent rabbit antisera. The purpose of this study was to evaluate the pulsed-field gel electrophoresis (PFGE) method for discrimination of Leptospira serovars.
Methods and Results:  Fourteen clinical isolates of Leptospira spp. were analysed by MAT before being characterized by PFGE. The isolates were compared with a library of 206 different reference Leptospira serovars. All the isolates gave clear profiles with high resolution. PFGE and MAT results were in agreement for all clinical isolates evaluated. Twelve isolates were classified as serovar Icterohaemorrhagiae/Copenhageni by PFGE. By MAT, these isolates were classified as serogroup Icterohaemorrhagiae with titres ranging from 3200 to 25 600. Two isolates were classified as serovar Canicola by PFGE, and as serogroup Canicola by MAT with titres higher than 3200.
Conclusions:  PFGE offers the advantages of simple, reliable and reproducible results.
Significance and Impact of the Study:  PFGE provides a convenient tool for the identification of clinical isolates.     

The homogeneity of vanB gene cluster among enterococcal isolates in Iran

Aims:  This study aimed to analyse the diversity of the vanB gene cluster in enterococcal species isolated from sewage treatment plants (STP) in Tehran, Iran.
Methods and Results:  The enterococcal isolates were collected from three sewage treatment plants in Tehran, Iran, during 2005. A total of 203 enterococcal isolates, collected over six rounds of sampling from three STPs were tested for the presence of vanB gene. Long-PCR showed that amongst the isolates, three Enterococcus faecium , one Enterococcus gallinarum and one Enterococcus casseliflavus harboured the complete vanB gene cluster.
Restriction fragment length polymorphism (RFLP) of the vanB1 gene cluster (5900 bp) from the isolates showed an identical pattern to a standard strain of Enterococcus faecalis (V583). None of the isolates were able to transfer the vanB gene in conjugation experiments. Different pulsed-field gel electrophoresis patterns were obtained for the three E. faecium isolates with vanB gene clusters .
Conclusions:  Our results indicated that the dissemination of vanB is not widespread in Tehran. Although only a few vanB positive isolates were detected, vanB was found in several enterococcal species.
Significance and Impact of the study:  In view of the lack of information on vanB resistance genes and their diversity in Iran, knowledge of the global dissemination of vanB genes in Enterococcus spp. is noteworthy.     

Spontaneous pathology of the baboon endocrine system

Background  Study of endocrine pathology in animal models is critical to understanding endocrine pathology in humans.
Methods  We evaluated 434 endocrine-related diagnoses from 4619 baboon necropsies, established the incidence of spontaneous endocrine pathology, and analyzed the clinical and biochemical data associated with the individual cases.
Results  The most common diagnoses in descending order, were pancreatic islet cell amyloidosis (n = 259), ovarian cysts (n = 50), pituitary adenoma (n = 37), pancreatic islet cell adenoma (n = 20), granulosa cell tumor (n = 15), thyroid adenoma (n = 11), adrenal hyperplasia (n = 10), thyroid carcinoma (n = 8), and pheochromocytoma (n = 6). The incidence of pancreatic islet cell amyloidosis progressively increased with age. Pheochromocytomas were associated with renal and heart failure. The incidence of pancreatic islet cell amyloidosis and adrenal pathology was similar to humans; the incidence of pituitary adenoma and thyroid pathology was lower than in humans.
Conclusions  Endocrine disease in baboons is common and shares clinical and biochemical characteristics with endocrine disease in humans.     

Auxin production by plant associated bacteria: impact on endogenous IAA content and growth of Triticum aestivum L.

Aims:  The aim of this study was to investigate the potential of bacterial strains of Bacillus, Pseudomonas, Escherichia, Micrococcus and Staphylococcus genera associated with wild herbaceous flora to enhance endogenous indole-3-acetic acid (IAA) content and growth of Triticum aestivum var. Inqalab-91.
Methods and Results:  Gas chromatography and mass spectrometric (GC–MS) analysis revealed that bacterial strains produced 0·6–8·22 μg IAA ml⁻¹ in the presence of L-tryptophan. Plant microbe experiments showed a significant positive correlation between auxin production by bacterial strains and endogenous IAA content of T. aestivum for GC–MS ( r  = 0·618; P  = 0.05) and colorimetric analysis ( r  = 0·693; P  = 0.01). Similarly, highly significant positive correlation for shoot length ( r  = 0·627; P  = 0.01) and shoot fresh weight ( r  = 0·626; P  = 0.01) was observed with auxin production under axenic conditions. Bacterial inoculations also enhanced shoot length (up to 29·16%), number of tillers (up to 97·35%), spike length (up to 25·20%) and seed weight (up to 13·70%) at final harvest.
Conclusions:  Bacterial strains have the ability to increase the endogenous IAA content and growth of T. aestivum var. Inqalab-91.
Significance and Impact of the Study:  Microbial strains of wild herbaceous flora can be effectively used to enhance the growth and yield of agronomically important crops.     

Serovar distribution and antimicrobial susceptibility of swine Salmonella isolates from clinically ill pigs in diagnostic submissions from Indiana in the United States

Aims:  To determine serovar distribution and levels of antimicrobial susceptibility of Salmonella isolated from clinically ill pigs in diagnostic submissions.
Methods and Results:  A total of 197 Salmonella isolates were obtained by the Indiana Animal Disease Diagnostic Laboratory from 2003 to 2005. Minimal inhibitory concentrations (MICs) were determined using the standard microbroth dilution method. The top four serovars identified were Salm. enterica serovar Typhimurium variant Copenhagen, Salm . Derby, Salm . Choleraesuis var. Kunzendorf and Salm . Typhimurium. All isolates were susceptible to the fluoroquinolones tested except that eight isolates were intermediate to difloxacin. The isolates showed a low prevalence of resistance to trimethoprim/sulphadiazine (Sxt), gentamicin (G), ceftiofur (Cf) and cephalothin (Cp) with low MIC₅₀ value of ≤0·5, 0·5, 1 and 4  μ g ml⁻¹, respectively. They showed a high prevalence of resistance to tetracycline (T; 83·8%), and a moderate prevalence to ampicillin (55·8%), spectinomycin (42·6%), ticarcillin (41·6%) and florfenicol (41·1%). There were more isolates of Salm . Typhimurium, including var. Copenhagen and Salm . Agona, that possessed multiple antimicrobial resistance to amoxicillin/clavulanic acid, ampicillin, ceftiofur and cephalothin (AxApCfCp) than the other serovars.
Conclusions:  The swine Salmonella isolates were susceptible to the fluoroquinolones, Sxt, G, Cf and Cp, but resistant to T.
Significance and Impact of the Study:  These findings provided useful information regarding antimicrobial susceptibility and resistance in dealing with clinical salmonellosis in pig herds.     

Identification and tracing of Enterococcus spp. by RAPD-PCR in traditional fermented sausages and meat environment

Aims:  Four local small-scale factories were studied to determine the sources of enterococci in traditional fermented sausages.
Methods and Results:  Different points during the production of a traditional fermented sausage type ( fuet ) were evaluated. Randomly amplified polymorphic DNA (RAPD)-PCR was used to type 596 Enterococcus isolates from the final products, the initial meat batter, the casing, the workers' hands and the equipment. Species-specific PCR-multiplex and the partial sequencing of atpA gene and 16S rRNA gene sequencing allowed the identification of the isolates: Enterococcus faecalis (31·4%), Enterococcus faecium (30·7%), Enterococcus sanguinicola (14·9%), Enterococcus devriesei (9·7%), Enterococcus malodoratus (7·2%), Enterococcus gilvus (1·0%), Enterococcus gallinarum (1·3%), Enterococcus casseliflavus (3·4%), Enterococcus hermanniensis (0·2%), and Enterococcus durans (0·2%) . A total of 92 different RAPD-PCR profiles were distributed among the different factories and samples evaluated. Most of the genotypes found in fuet samples were traced back to their source.
Conclusions:  The major sources of enterococci in the traditional fermented sausages studied were mainly the equipment followed by the raw ingredients, although a low proportion was traced back to human origin.
Significance and Impact of the Study:  This work contributes to determine the source of enterococcal contamination in fermented sausages and also to the knowledge of the meat environment.     

Free radical scavenging activity of Lactobacillus fermentum in vitro and its antioxidative effect on growing–finishing pigs

Aims:  To evaluate the free radical-scavenging capacity of Lactobacillus fermentum and its effects on antioxidant enzyme levels in finishing pigs.
Methods and Results:  The free radical-scavenging activity of Lact. fermentum was analysed in vitro . The tested Lactobacillus showed a high scavenging ability against DPPH (1,1-diphenyl-2-picrylhydrazyl), superoxide and hydroxyl radicals which was dose dependent. Subsequently, 108 crossbred pigs weighing 20·67 BW, were allotted to dietary treatments including a basal diet or the basal diet supplemented with either aureomycin or 10·2 × 10⁷  Lact. fermentum CFU g⁻¹ diet. Supplementation of Lact. fermentum increased total antioxidant capacity ( P  < 0·01) in serum from 50 kg pigs, while serum superoxide dismutase ( P  = 0·01) and glutathione peroxidase ( P  < 0·01) increased, and malondialdehyde levels decreased ( P  < 0·01) in 90 kg pigs. Hepatic catalase ( P  = 0·04), muscle superoxide dismutase ( P  < 0·01) and copper–zinc-superoxide dismutase were enhanced ( P  = 0·01), whereas malondialdehyde levels were reduced ( P  = 0·05) by Lact. fermentum .
Conclusions:  The free radical-scavenging capacity of Lact. fermentum was dose dependent and its supplementation improved the antioxidant status of pigs.
Significance and Impact of the Study:  Lactobacillus fermentum could be used to alleviate oxidative stress and increase pig performance and improve pork quality.     

Comparison of methods to detect resistance of Penicillium expansum to thiabendazole

Aims:  Because of the lack of a standard method, the aim of this work is to evaluate the suitability of the broth microdilution method CLSI M38-A in determining the resistance level of some Penicillium expansum isolates to thiabendazole (TBZ). The ability of the isolates to produce patulin (PAT) and citrinin (CIT) has been also assessed.
Methods and Results:  Penicillium expansum isolates (128) were assayed (apples, pears, grapes and five reference strains). It was observed that 69·4% of the strains isolated from apples and pears were resistant to TBZ. Sensitive isolates were inhibited at 0·25–0·5 μg ml⁻¹ whilst resistant isolates still grew at 512 μg ml⁻¹. PAT was produced by all P. expansum isolates. CIT was detected in 98·8% of TBZ-resistant isolates and in 89·1% of the TBZ-sensitive isolates.
Conclusions:  The preliminary screening method combined with the adaptation of the method CLSI M38-A, can be a good strategy to be used in assessing the in vitro activity of TBZ against a large number of isolates.
Significance and Impact of the Study:  The proposed methodology can be a contribution to the standardization of susceptibility tests to fungicides against P. expansum.     

Influence of body condition and forage type on prevalence of Escherichia coli O157:H7 and Salmonella in grazing beef cows

Aim:  To determine the influence of body condition (BC) and forage type on the prevalence of faecal shedding of Escherichia coli O157:H7 and Salmonella from beef cows.
Methods and Results:  Thin or moderately conditioned cows ( n  =   115) were randomly assigned to graze either common bermudagrass ( n  =   3 pastures) or toxic endophyte-infected tall fescue ( n  =   3 pastures) for 62 days. Faecal samples were collected on day 0, 30 and 62. Overall percentage of faecal samples positive for E. coli O157:H7 was 2·6% and 2·0% for Salmonella . Percentage of cows positive for both E. coli O157:H7 and Salmonella on at least one occasion was 6·1%. BC, forage type or the interaction did not influence the prevalence of E. coli O157:H7 or Salmonella in the faeces of cows.
Conclusions:  BC at initiation of the grazing period or loss of BC in moderate conditioned cows during the grazing period did not influence faecal shedding of E. coli O157:H7 or Salmonella . Consumption of either forage type did not influence faecal shedding of either E. coli O157:H7 or Salmonella in beef cows of thin or moderate BC.
Significance and Impact of the Study:  Change in BC that typically occurs during the normal production cycle in grazing cows did not influence faecal shedding of pathogenic bacteria regardless of forage type.     

Viable ultramicrocells in drinking water

Aims:  To examine the diversity of cultivable 0·2 micron filtrate biofilm forming bacteria from drinking water systems.
Methods and Results:  Potable chlorinated drinking water hosts phylogenetically diverse ultramicrocells (UMC) (0·2 and 0·1  μ m filterable). UMC (starved or dwarf bacteria) were isolated by cultivation on minimal medium from a flow system wall model with polyvinyl chloride (PVC) pipes. All cultivated cells (25 different isolates) did not maintain their ultra-size after passages on rich media. Cultured UMC were identified by their 16S ribosomal DNA sequences. The results showed that they were closely related to uncultured and cultured members of the Proteobacteria, Actinobacteria and Firmicutes. The isolates of phylum Actinobacteria included representatives of a diverse set of Actinobacterial families: Micrococcaceae, Microbacteriaceae, Dermabacteraceae, Nocardiaceae and Nocardioidaceae.
Conclusions:  This study is the first to show an abundance of cultivable UMC of various phyla in drinking water system, including a high frequency of bacteria known to be involved in opportunistic infections, such as Stenotrophomonas maltophilia, Microbacterium sp., Pandoraea sp. and Afipia strains.
Significance and Impact of the Study:  Chlorinated tap water filtrate (0·2 and 0·1  μ m) still harbours opportunistic micro-organisms that can pose some health threat.     

Comparison of Lactococcus garvieae strains isolated in northern Italy from dairy products and fishes through molecular typing

Aims:  To investigate the genetic relatedness between Lactococcus garvieae strains isolated from fish and dairy samples collected in northern Italy, using random-amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), Sau -PCR and amplified fragment length polymorphism (AFLP).
Methods and Results:  Eighty-one isolates from bovine and caprine dairy products ( n  = 53) and from diseased rainbow trouts and other fishes ( n  = 28) were examined. All methods showed a typeability of 100%, repeatability ranging from 84·4% to 97·5% and discriminatory powers from 0·798 to 0·986. Dairy and fish strains revealed a low genetic relatedness as they are often grouped into distinct clusters. RAPD analysis discriminated 52 genotypes when primer M13 was used, whereas with primer P5 only 27 genotypes were identified. When Sau -PCR was performed, 13 genotypes were detected while AFLP analysis allowed the differentiation of 32 genotypes.
Conclusion:  L. garvieae strains isolated from dairy samples are generally not related to those collected from fish lactococcosis outbreaks.
Significance and Impact of the Study:  L. garvieae strains exhibit a genetic diversity related to the specific animal host they colonize. RAPD M13 fingerprinting proved to be a molecular tool for comparing isolates, whereas Sau -PCR and AFLP analyses were useful techniques to investigate the distribution of L. garvieae populations in the environment.     

Modelling the growth of Weissella cibaria as a function of fermentation conditions

Aims:  To investigate the effect of pH, water activity ( a _w) and temperature on the growth of Weissella cibaria DBPZ1006, a lactic acid bacterium isolated from sourdoughs.
Methods and Results:  The kinetics of growth of W. cibaria DBPZ1006 was investigated during batch fermentations as a function of pH (4·0–8·0), a _w (0·935–0·994) and temperature (10–45°C) in a rich medium. The growth curve parameters (lag time, growth rate and asymptote) were estimated using the dynamic model of Baranyi and Roberts (1994. A dynamic approach to predicting bacterial growth in food. Int J Food Microbiol 23, 277–294). The effect of pH, a _w and temperature on maximum specific growth rate (μ_max) were estimated by fitting a cardinal model. μ_max under optimal conditions (pH = 6·6, a _w = 0·994, T  = 36·3°C) was estimated to be 0·93 h⁻¹. Minimum and maximum estimated pH and temperature for growth were 3·6 and 8·15, and 9·0°C and 47·8°C, respectively, while minimum a _w was 0·918 (equivalent to 12·2% w/v NaCl).
Conclusions:  Weissella cibaria DBPZ1006 is a fast-growing heterofermentative strain, which could be used in a mixed starter culture for making bread.
Significance and Impact of the Study:  This is the first study reporting the modelling of the growth of W. cibaria , a species that is increasingly being used as a starter in sourdough and vegetable fermentations.     

Characterization of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis from eastern China

Aims:  The aim of this study was to investigate the features of rpoB gene mutations associated with Rifampin (RIF) resistance in Mycobacterium tuberculosis ( M. tuberculosis ) in eastern China.
Methods and Results:  The mutations of rpoB gene in 56 clinical isolates of M. tuberculosis resisted to one to four first-line drugs (rifampin, isonicotinyl hydrazide, ethambutol and streptomycin) were analysed by polymerase chain reaction single strand conformation polymorphism analysis (PCR-SSCP) and DNA sequencing. The results of PCR-SSCP showed 52 isolates were positive (existing rpoB mutation) including 47 isolates resisted to RIF. Subsequent results of DNA sequencing showed that 54 isolates had rpoB gene mutation including 49 isolates resisted to RIF. The most frequently mutated sites were at codons 526 (73·2%), 513 (10·7%) and 531 (3·5%).
Conclusions:  The rpoB codon 526 was the most frequently mutated site of RIF-resistant M. tuberculosis strains in eastern China and its frequency is significantly higher ( P  < 0·0001) compared with that in other areas of China and in other geographic regions worldwide.
Significance and Impact of the Study:  Our results reveal that geographic variation is responsible for rpoB mutations in M. tuberculosis and the resulting information will be helpful to improve a novel rapid molecular drug resistance screening approach for MDR TB.     

Antimicrobial resistance and in vitro gene transfer in bacteria isolated from the ulcers of EUS-affected fish in India

Aims:  The occurrence of drug resistance and plasmid-mediated transferability was investigated in 15 Aeromonas isolates collected from the ulcers of epizootic ulcerative syndrome (EUS)-affected fishes Katla ( Catla catla ), Mrigel ( Cirrhinus mrigala ) and Punti ( Puntius sp.).
Methods and Results:  Disc diffusion assay showed that all the strains were resistant to ampicillin and sensitive to streptomycin. Of the 15 isolates examined, 93·3% isolates were resistant to erythromycin, sulfadiazine and novobiocin, while 66% were resistant to rifampin and 20% to chloramphenicol. All isolates harboured plasmids with sizes ranging from 64 to 23 kbp with a 23-kbp plasmid in common. Plasmids from 11 Aeromonas strains were transferred to Escherichia coli DH5α recipient strain along with the transfer of ampicillin, erythromycin and chloramphenicol resistance determinants with frequencies ranging from 7·0 × 10⁻⁶ to 1·8 × 10⁻⁵ transconjugants per recipient cell.
Conclusions:  The resistance to ampicillin, erythromycin, sulfadiazine, novobiocin and chloramphenicol is prevalent among the bacteria isolated from EUS-affected fish, and resistant determinants of some of these antibiotics have been transferred to the bacteria of other origin.
Significance and Impact of the Study:  The emergence of antibiotic resistance bacteria and gene transfer in vitr o suggests that antibiotics should be used more cautiously to treat Aeromonas infections in aquaculture.