Incidence of lysogeny, colicinogeny, and drug resistance in enterobacteria isolated from sewage and from rectum of humans and some domesticated species.

Enterobacteria were isolated by streaking swabs of sewage and rectal swabs from human volunteers from domesticated animals. Thirty strains of human origin were identified as Escherichia coli. Out of 1,367 rectal isolates of animal origin, 21% were lysogenic (phi+), 29% were colicinogenic (col+), and 7% were col+ phi+. Out of 85 rectal samples more than 60% harbored variable numbers of col+ or phi+ bacteria. Lysogens harboring homoimmune prophages were detectable in six out of eight human subjects in sequential samples taken at weekly intervals. Chickens in Hong Kong are fed on antibiotic-containing feeds; the avian isolates contained the highest frequency (98%) of drug-resistant bacteria, whereas only 39% of the bovine and 61% of the human isolates were drug resistant. Transmissible drug resistance was demonstrable in sewage isolates and those from animal sources; the highest frequency (58%) of resistance donors was shown by the avian isolates, and the lowest (9%) was shown by the bovine isolates. Unselected marker analysis has shown that a vast majority of multiply resistant donors of diverse origins are able to transmit multiple resistance.     

Evaluation of the specificity of Salmonella PCR primers using various intestinal bacterial species

AIMS: Nine sets of PCR primers targeting Salmonella were evaluated for their specificity with pure cultures of intestinal-associated bacteria prior to their application to Salmonella detection in faecal samples. METHODS AND RESULTS: Gene targets of PCR primers included: 16S rDNA, a Salmonella pathogenicity island I virulence gene, Salmonella enterotoxin gene (stn), invA gene, Fur-regulated gene, histidine transport operon, junction between SipB and SipC virulence genes, Salmonella-specific repetitive DNA fragment, and multiplex targeting invA gene and spvC gene of the virulence plasmid. Fifty-two Salmonella strains were used to determine sensitivity; five strains from related genera and 45 intestinal bacteria were used to evaluate specificity. All primers amplified DNA from Salmonella strains, although two primer sets failed to amplify Salmonella DNA from either Salmonella bongori (hilA) or subgroups VI or VII (16S rDNA). There was no detected amplification of DNA from related bacterial genera with any of nine PCR assays. Six of the PCR assays amplified DNA for some intestinal bacteria. CONCLUSIONS: Only three primer pairs were determined to be suitable for application of PCR amplification of Salmonella in faecal samples - 16S rDNA, stn and histidine transport operon. We are currently evaluating their sensitivity of detection of Salmonella in faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the importance of internal lab validation of PCR primers prior to application to the type of samples of interest. Information from this evaluation can be applied in other labs to facilitate choosing Salmonella PCR primers.     

Antibiotic Resistance in Food-Borne Bacterial Contaminants in Vietnam

This study was conducted to examine the rate of contamination and the molecular characteristics of enteric bacteria isolated from a selection of food sources in Vietnam. One hundred eighty raw food samples were tested; 60.8% of meat samples and 18.0% of shellfish samples were contaminated with Salmonella spp., and more than 90% of all food sources contained Escherichia coli. The isolates were screened for antibiotic resistance against 15 antibiotics, and 50.5% of Salmonella isolates and 83.8% of E. coli isolates were resistant to at least one antibiotic. Isolates were examined for the presence of mobile genetic elements conferring antibiotic resistance. Fifty-seven percent of E. coli and 13% of Salmonella isolates were found to contain integrons, and some isolates contained two integrons. Sequencing results revealed that the integrons harbored various gene cassettes, including aadA1, aadA2, and aadA5 (resistance to streptomycin and spectinomycin), aacA4 (resistance to aminoglycosides), the dihydrofolate reductase gene cassettes dhfrXII, dfrA1, and dhfrA17 (trimethoprim resistance), the beta-lactamase gene bla_PSE1 (ampicillin resistance), and catB3 (chloramphenicol resistance). Plasmids were also detected in all 23 antibiotic-resistant Salmonella isolates and in 33 E. coli isolates. Thirty-five percent of the Salmonella isolates and 76% of the E. coli isolates contained plasmids of more than 95 kb, and some of the isolates contained two large plasmids. Conjugation experiments showed the successful transfer of all or part of the antibiotic resistance phenotypes among the Salmonella and E. coli food isolates. Our results show that enteric bacteria in raw food samples from Vietnam contain a pool of mobile genetic elements and that the transfer of antibiotic resistance can readily occur between similar bacteria.     

The identification of a tetracycline resistance gene tet(M), on a Tn916-like transposon,in the Bacillus cereus group

In order to investigate whether resistance genes present in bacteria in manure could transfer to indigenous soil bacteria, resistant isolates belonging to the Bacillus cereus group (Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis) were isolated from farm soil (72 isolates) and manure (12 isolates) samples. These isolates were screened for tetracycline resistance genes (tet(K), tet(L), tet(M), tet(O), tet(S) and tet(T)). Of 88 isolates examined, three (3.4%) isolates carried both tet(M) and tet(L) genes, while four (4.5%) isolates carried the tet(L) gene. Eighty-one (92.1%) isolates did not contain any of the tested genes. All tet(M) positive isolates carried transposon Tn916 and could transfer this mobile DNA element to other Gram-positive bacteria.     

Development of a combined selective enrichment method and polymerase chain reaction (PCR) assay for sensitive detection of Salmonella in food samples

PCR assays were formatted using primer pairs homologous to phoE and invA genes. The amplification conditions were optimized with pure cultures and reactions were carried out to define selectivity, specificity and sensitivity of both primer pairs. The performance of the invA primer pair was better than that of the phoE pair, making the specific detection of Salmonella serovars and strains isolated from different food samples possible. Using the invA primer pair, the combined selective enrichment method with the polymerase chain reaction assay was established and used to detect Salmonella from artificially multi-contaminated food samples. The complete procedure detected as few as three cells of Salmonella (3 c.f.u.) from milk and meat samples.     

Virulence determinants invA and spvC in salmonellae isolated from poultry products, wastewater, and human sources.

The presence of two virulence foci, invA and spvC, in Salmonella isolates obtained from poultry, wastewater, and human sources was determined. All isolates (n = 245) were positive for the invA gene sequence. Differences in degree of invasiveness were apparent with the Madin Darby canine kidney cell line, as only 79 of 159 randomly selected isolates (49.7%) tested were invasive at > 0.1% of the inoculum. 25% were invasive between 0.1 and 1.0% of the inoculum, and 24.5% were invasive at > 1.0% of the inoculum. There was a significant correlation between degree of invasion and source from which the isolate was recovered but no correlation between geographic origin of poultry isolates and degree of invasion. Only 37 of 245 isolates (15.1%) hybridized with the spvC DNA probe. All isolates that were recovered from a commercial egg production environment and chicken eggs and whose sequences exhibited homology with the spvC gene sequence were determined to be either Salmonella enteritidis PT 23 or PT 13. The sequences of few isolates from ceca and none from wastewater or humans demonstrated homology with the spvC gene.     

Bacterial contaminants and their antibiotic susceptibility patterns in ready-to-eat foods vended in Ogun state,Nigeria

Contamination of ready-to-eat (RTE) foods by pathogenic bacteria may predispose consumers to foodborne diseases. This study investigated the presence of bacterial contaminants and their antibiotic susceptibility patterns in three locally processed RTE foods (eko, fufu and zobo) vended in urban markets in Ogun state, Nigeria. Bacteria isolated from a total of 120 RTE food samples were identified by 16S rRNA gene phylogeny while susceptibility patterns to eight classes of antibiotics were determined by the disc diffusion method. Species belonging to the genera Acinetobacter and Enterobacter were recovered from all RTE food types investigated, Klebsiella and Staphylococcus were recovered from eko and fufu samples, while those of Shigella were recovered from eko samples. Enterobacter hormaechei was the most prevalent species in all three RTE food types. Precisely 99% of 149 isolates were multidrug-resistant, suggesting a high risk for RTE food handlers and consumers. Co-resistance to ampicillin and cephalothin was the most frequently observed resistance phenotype. Results demonstrate that improved hygiene practices by food processors and vendors are urgently required during RTE processing and retail. Also, adequate food safety guidelines, regulation and enforcement by relevant government agencies are needed to improve the safety of RTE foods and ensure the protection of consumer health.     

Antibiotic susceptibility patterns and resistance genes of starter cultures and probiotic bacteria used in food

A survey of starter and probiotic cultures was carried out to determine the current antibiotic resistance situation in microbial food additives in Switzerland. Two hundred isolates from 90 different sources were typed by molecular and other methods to belong to the genera Lactobacillus (74 samples), Staphylococcus (33 samples), Bifidobacterium (6 samples), Pediococcus (5 samples), or were categorized as lactococci or streptococci (82 samples). They were screened for phenotypic resistances to 20 antibiotics by the disk diffusion method. Twenty-seven isolates exhibiting resistances that are not an intrinsic feature of the respective genera were further analyzed by microarray hybridization as a tool to trace back phenotypic resistances to specific genetic determinants. Their presence was finally verified by PCR amplification or Southern hybridization. These studies resulted in the detection of the tetracycline resistance gene tet(K) in 5 Staphylococcus isolates used as meat starter cultures, the tetracycline resistance gene tet(W) in the probiotic cultures Bifidobacterium lactis DSM 10140 and Lactobacillus reuteri SD 2112 (residing on a plasmid), and the lincosamide resistance gene lnu(A) (formerly linA) in L. reuteri SD 2112.     

重组酶介导等温核酸扩增方法快速检测土壤沙门氏菌

【背景】沙门氏菌(Salmonella)是一种可以引起人畜患病的致病菌,也是最主要的食源性细菌之一。土壤中的沙门氏菌可通过蔬菜等植物进入人体,引发食物中毒。但由于土壤性质及其他微生物的干扰,如何快速甄别土壤是否受到沙门氏菌的污染仍是一个难题。【目的】建立一种快速、灵敏检测土壤沙门氏菌的实时重组酶介导等温核酸扩增(Real-Time Recombinase Aided Amplification,RT-RAA)方法。【方法】针对沙门氏菌invA基因序列设计特异性引物和探针,构建含有invA基因待检片段的重组质粒,评价RT-RAA方法的灵敏度;分别以肠炎沙门氏菌、大肠杆菌、福氏志贺氏菌和金黄色葡萄球菌的基因组DNA为模板,评价RT-RAA方法的特异性;RT-RAA方法用于番茄、生姜土壤中沙门氏菌的检测,同时用平板培养法进行验证。【结果】RT-RAA方法可用于重组质粒中invA基因片段的检测,在39℃条件下,20 min内即可获得检测结果,最低检测质粒拷贝数为10拷贝/反应,而且与大肠杆菌、福氏志贺氏菌和金黄色葡萄球菌无交叉反应。土壤样品DNA的RT-RAA检测结果显示,供试番茄土已被沙门氏菌污染,而生姜土则没有,与平板培养结果一致。【结论】RT-RAA方法具有灵敏度高和特异性强的特点,可用于土壤沙门氏菌污染的快速检测。     

Mass Producing Food Traditions for West Africans Abroad

In this article, I examine West African foods sold mainly in specialty grocery stores, focusing on how technologies used in food production in West Africa are referenced in the brand names and packaging of processed African foods sold in the United States. Through their association with "timeless" West African food-processing techniques, such foods evoke memories of childhood and home. Yet the transformation of West African foods through new technologies of processing, packaging, and branding reflects different time and health concerns of West African immigrants living in the United States. Through their purchase of time-saving, mass-produced, and hygienically packaged foodstuffs, which are ideologically similar to but technologically very different from the production processes and cooking in Africa, West Africans in the United States use food to maintain social relations with their particular families, hometown associations, and religious groups, while also constituting national, regional, and global connections through the reinvention of food traditions.     

Diversity of tetracycline resistance genes in bacteria from aquaculture sources in Australia

AIMS: To determine the genetic determinants responsible for tetracycline resistance in oxytetracycline resistant bacteria from aquaculture sources in Australia. METHODS AND RESULTS: Twenty of 104 (19%) isolates tested were resistant to oxytetracycline (MIC > or = 16 microg ml(-1)). Using polymerase chain reaction (PCR) amplification, one or more tet genes were detected in 15/20 (75%) isolates tested, but none were found in 5/20 (25%). tetM (50%) was the most common determinant, followed by tetE (45%), tetA (35%) and tetD (15%). Five of 12 oxytetracycline resistant isolates studied were able to transfer their R-plasmid to Escherichia coli recipients of chicken, pig and human origin. tetA, tetD and tetM were found to be transferred while tetE was not transferred. Southern hybridization and PCR were used to confirm transfer of determinants. CONCLUSIONS: Bacterial isolates from aquaculture sources in Australia harbour a variety of tetracycline resistance genes, which can be transferred to other bacteria of different origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteria from aquaculture sources in Australia contribute to the resistance gene pool reservoir. The in vitro transfer of tetracycline R-plasmid from aquatic bacteria to E. coli isolates from various sources is an indication of the potential public health risk associated with these resistance determinants.     

Diverse Gene Cassettes in Class 1 Integrons of Facultative Oligotrophic Bacteria of River Mahananda,West Bengal,India

Background

In this study a large random collection (n = 2188) of facultative oligotrophic bacteria, from 90 water samples gathered in three consecutive years (2007–2009) from three different sampling sites of River Mahananda in Siliguri, West Bengal, India, were investigated for the presence of class 1 integrons and sequences of the amplification products.

Methodology/Principal Findings

Replica plating method was employed for determining the antibiotic resistance profile of the randomly assorted facultative oligotrophic isolates. Genomic DNA from each isolate was analyzed by PCR for the presence of class 1 integron. Amplicons were cloned and sequenced. Numerical taxonomy and 16S rRNA gene sequence analyses were done to ascertain putative genera of the class 1 integron bearing isolates. Out of 2188 isolates, 1667 (76.19%) were antibiotic-resistant comprising of both single-antibiotic resistance (SAR) and multiple-antibiotic resistant (MAR), and 521 (23.81%) were sensitive to all twelve different antibiotics used in this study. Ninety out of 2188 isolates produced amplicon(s) of varying sizes from 0.15 to 3.45 KB. Chi-square (χ²) test revealed that the possession of class 1 integron in sensitive, SAR and MAR is not equally probable at the 1% level of significance. Diverse antibiotic-resistance gene cassettes, aadA1, aadA2, aadA4, aadA5, dfrA1, dfrA5, dfrA7, dfrA12, dfrA16, dfrA17, dfrA28, dfrA30, dfr-IIe, blaIMP-9, aacA4, Ac-6′-Ib, oxa1, oxa10 and arr2 were detected in 64 isolates. The novel cassettes encoding proteins unrelated to any known antibiotic resistance gene function were identified in 26 isolates. Antibiotic-sensitive isolates have a greater propensity to carry gene cassettes unrelated to known antibiotic-resistance genes. The integron-positive isolates under the class Betaproteobacteria comprised of only two genera, Comamonas and Acidovorax of family Comamonadaceae, while isolates under class Gammaproteobacteria fell under the families, Moraxellaceae, Pseudomonadaceae, Aeromonadaceae and Enterobacteriaceae.

Conclusions

Oligotrophic bacteria are good sources of novel genes as well as potential reservoirs of antibiotic resistance gene casettes.     

Effect of the growth promoter avilamycin on emergence and persistence of antimicrobial resistance in enteric bacteria in the pig

AIM: To assess the effect of the growth promoter avilamycin on emergence and persistence of resistance in enteric bacteria in the pig. METHODS AND RESULTS: Pigs (treated with avilamycin for 3 months and controls) were challenged with multi-resistant Salmonella Typhimurium DT104 and faecal counts were performed for enterococci, Escherichia coli, S. Typhimurium and Campylobacter (before, during and 5 weeks post-treatment). Representative isolates were tested for antibiotic resistance and for the presence of resistance genes. Avilamycin-resistant Enterococci faecalis (speciated by PCR) were isolated from the treated pigs and continued to be detected for the first week after treatment had ceased. The avilamycin-resistance gene was characterized by PCR as the emtA gene and speciation by PCR. MIC profiling confirmed that more than one strain of Ent. faecalis carried this gene. There was no evidence of increased antimicrobial resistance in the E. coli, Salmonella and Campylobacter populations, although there was a higher incidence of tetB positive E. coli in the treated pigs than the controls. CONCLUSION: Although avilamycin selects for resistance in the native enterococci population of the pig, no resistant isolates were detected beyond 1 week post-treatment. This suggests that resistant isolates were unable to persist once selective pressure was removed and were out-competed by the sensitive microflora. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest the risk of resistant isolates becoming carcass contaminants and infecting humans could be minimized by introducing a withdrawal period after using avilamycin and prior to slaughter.     

Quantitative determination of free-DNA uptake in river bacteria at the single-cell level by in situ rolling-circle amplification

Detection of plasmid DNA uptake in river bacteria at the single-cell level was carried out by rolling-circle amplification (RCA). Uptake of a plasmid containing the green fluorescent protein gene (gfp) by indigenous bacteria from two rivers in Osaka, Japan, was monitored for 506 h using this in situ gene amplification technique with optimized cell permeabilization conditions. Plasmid uptake determined by in situ RCA was compared to direct counts of cells expressing gfp under fluorescence microscopy to examine differences in detection sensitivities between the two methods. Detection of DNA uptake as monitored by in situ RCA was 20 times higher at maximum than that by direct counting of gfp-expressing cells. In situ RCA could detect bacteria taking up the plasmid in several samples in which no gfp-expressing cells were apparent, indicating that in situ gene amplification techniques can be used to determine accurate rates of extracellular DNA uptake by indigenous bacteria in aquatic environments.     

Food commensal microbes as a potentially important avenue in transmitting antibiotic resistance genes

The rapid emergence of antibiotic-resistant (ART) pathogens is a major threat to public health. While the surfacing of ART food-borne pathogens is alarming, the magnitude of the antibiotic resistance (AR) gene pool in food-borne commensal microbes is yet to be revealed. Incidence of ART commensals in selected retail food products was examined in this study. The presence of 10(2)-10(7) CFU of ART bacteria per gram of foods in many samples, particularly in ready-to-eat, 'healthy' food items, indicates that the ART bacteria are abundant in the food chain. AR-encoding genes were detected in ART isolates, and Streptococcus thermophilus was found to be a major host for AR genes in cheese microbiota. Lactococcus lactis and Leuconostoc sp. isolates were also found carrying AR genes. The data indicate that food could be an important avenue for ART bacterial evolution and dissemination. AR-encoding plasmids from several food-borne commensals were transmitted to Streptococcus mutans via natural gene transformation under laboratory conditions, suggesting the possible transfer of AR genes from food commensals to human residential bacteria via horizontal gene transfer.     

Polymerase chain reaction analysis of mitochondrial DNA polymorphism in N'Dama and Zebu cattle

A study of polymorphisms of mitochondrial DNA (mtDNA) of West African N'Dama (Bos taurus) and East African Zebu (B. indicus) cattle was carried out to obtain information on maternal phylogenetic relationships between these breeds. A relatively large sample size was made possible by using polymerase chain reaction (PCR) amplification of DNA prepared from small blood samples to generate fragments of two known polymorphic mtDNA regions, one within the gene encoding subunit 5 of NADH dehydrogenase and one encompassing the entire D-loop. This approach allowed us to achieve a higher resolution restriction analysis on mtDNA from more animals than would have been possible by conventional methods. PCR-amplified mtDNA of 58 animals from five populations was examined at 26 restriction sites by 16 enzymes. In this way 154 nucleotides of mtDNA were scanned for polymorphism. Six polymorphic sites were located by this means, five of which were within the D-loop and one of which was within the NADH dehydrogenase 5 gene. None of the polymorphisms observed could be con sidered typical of breed or type.     

Homology of Escherichia coli R773 arsA, arsB, and arsC genes in arsenic-resistant bacteria isolated from raw sewage and arsenic-enriched creek waters.

The occurrence and diversity of the Escherichia coli R773 ars operon were investigated among arsenic-resistant enteric and nonenteric bacteria isolated from raw sewage and arsenic-enriched creek waters. Selected isolates from each creek location were screened for ars genes by colony hybridization and PCR. The occurrence of arsA, arsB, and arsC determined by low-stringency colony hybridization (31 to 53% estimated mismatch) was 81, 87, and 86%, respectively, for 84 bacteria isolated on arsenate- and arsenite-amended media from three locations. At moderate stringency (21 to 36% estimated mismatch), the occurrence decreased to 42, 56, and 63% for arsA, arsB, and arsC, respectively. PCR results showed that the ars operon is conserved in some enteric bacteria isolated from creek waters and raw sewage. The occurrence of the arsBC genotype was about 50% in raw sewage enteric bacteria, while arsA was detected in only 9.4% of the isolates (n = 32). The arsABC and arsBC genotypes occurred more frequently in enteric bacteria isolated from creek samples: 71.4 and 85.7% (n = 7), respectively. Average sequence divergence within arsB for six creek enteric bacteria was 20% compared to that of the E. coli R773 ars operon. Only 1 of 11 pseudomonads screened by PCR was positive for arsB. The results from this study suggest that significant divergence has occurred in the ars operon among As-resistant E. coli strains and in Pseudomonas spp.     

Prevalence, distribution and antibiotic resistance pattern among enterococci species in two traditional fermented dairy foods

The prevalence, distributions and antibiotic resistance pattern among enterococci species were determined. A total of 30 samples of Nigerian traditional fermented dairy food were positive to presence of enterococci, with viable counts of 4.17 log CFU/g in nunu, lower than 4.55 log CFU/g observed in wara samples. Twenty-five representative strains were characterized by a combination of phenotypic and genomic typing based on 16S rRNA gene and multi-locus sequencing analysis (MLSA) of RNA polymerase A (rpoA) and phenylanaline synthase (pheS) genes sequencing; these strains were identified as Enterococcus faecium (84 %) and Enterococcus faecalis (16 %). All the 95 enterococci isolated from wara and nunu samples were alpha haemolytic with multi-drug resistance to 10 antimicrobials regardless of class. Four strains were sensitive to chloramphenicol (30 μg) while 33.7 % of the total isolates were resistant to vancomycin from 5 μg. This information will enhance understanding of Enterococcus drug resistance and distribution in traditional fermented foods to support safety and guarantee quality of traditional foods in West Africa.     

Intercontinental variation of Sclerospora graminicola

Collections of S. graminicola were made from local pearl millet crops in West Africa and India. Asexual inoculum was derived from the collections in Polythene tunnels and used to infect pearl millet cultivars from both continents. Analysis of deviance of the incidence of disease was performed on the logit scale using GLIM. The host × pathogen interactions were interpreted by use of the Finlay-Wilkinson regression technique widely used in the study of genotype × environment interactions. While the West African collections were consistently more pathogenic than the Indian ones, West African hosts were potentially more susceptible to Indian than to West African pathogen isolates; conversely some Indian hosts were more vulnerable to West African than to Indian isolates. There was a fairly stable relationship between incidence of disease and relative pathogenicity of West African isolates but the Indian isolates were more variable in their susceptibility to changing background levels of disease. These results could influence resistance breeding strategies in pearl millet improvement programmes. The methods of analysis could be valuable for application to other complex and ill-defined pathosystems.