Molecular identification and anti-pathogenic activities of putative probiotic bacteria isolated from faeces of healthy elderly individuals

One hundred and nine lactic acid bacterial strains (56 bifidobacteria-like and 53 lactobacilli-like) were isolated from faecal samples donated by healthy elderly individuals (>65 years old). Isolates were identified to species level by phenotypic analysis (by API) and by 16S rDNA sequencing. Eleven species of Lactobacillus and six species of Bifidobacterium were identified. The most frequently isolated lactobacillus was L. fermentum and the most frequently isolated bifidobacterium was closely related to B. infantis by 16S rDNA sequence alignment. The isolates were characterized for their antimicrobial activity against Clostridium difficile, enteropathogenic Escherichia coli (EPEC), verocytotoxigenic E. coli (VTEC) and Campylobacter jejuni. The lactobacilli displayed variations in their antimicrobial activity with few strains showing inhibitory activity against all pathogens. The bifidobacteria displayed higher levels of inhibitory activity against C. jejuni and Cl. difficile than against the E. coli strains. Keywords: Lactobacillus, Bifidobacterium, elderly, gastrointestinal microbiota, inhibition, Clostridium difficile, enteropathogenic Escherichia coli (EPEC), verocytotoxigenic E. coli (VTEC), Campylobacter jejuni.

Rodents as carriers of disease: preliminary studies in the United Kingdom

The University of Reading has conducted some preliminary work on the prevalence of Campylobacter spp., Salmonella spp. and Arenavirus in Norway rats trapped from farms and semi-urban areas in central southern England. Campylobacter is the cause of a notificable disease in the UK, with 57,772 cases reported for England and Wales in 2009. Transmission to humans is believed to be primarily through undercooked meat, from contaminated water, and through contact with pets; and symptoms include a high temperature, severe diarrhoea, vomiting and abdominal pain. Ninety-seven per-cent of sporadic cases have been attributed to farm animals, and in particular the meat and poultry industry. There are eighteen species of Campylobacter, eleven of which can be pathogenic to humans; although the principal species that cause gastrointestinal disease in humans are C. jejuni and C. coli; although C. lari, C. helveticus and C. upsaliensis are also involved. Salmonella species also causes a gastrointestinal disease, and in the UK, is common in chicken and has been linked to egg production. Species are typed using antigen specific agglutination tests, or by their susceptibility to specific bacteriophage. Some strains are known to be linked with human disease (eg. S. enteritidis PT4).

The role of rodents as carriers of disease on UK farms: a preliminary investigation

In the UK, Campylobacter spp. and Lymphocytic Choriomeningitis Virus (LCMV), an Old World arenavirus, cause two zoonoses of concern that may be transmissible from rodents to humans and livestock. The aims of this preliminary investigation were to examine the occurrence of Campylobacter spp. and LCMV in Norway rats Rattus norvegicus on UK farms and to identify and characterise the Sequence Types of the Campylobacter isolates. Samples were collected from wild Norway rats and fresh Norway rat faeces. Multi Locus Sequence Typing (MLST) was performed on C. spp. isolates and samples were tested for arenavirus RNA by RT-PCR. Six C. spp. isolates were identified. One isolate was C. lari and five isolates were C. jejuni. Following MSLT profiling, three unique C. jejuni sequence types were identified. Two of which are novel and the third is typically associated with livestock and human infection. Nine positive results for LCMV were obtained giving an overall prevalence of 25% across four sites. This is higher than previously reported for this species.

Restriction fragment length polymorphism of polymerase chain reaction products applied to the differentiation of poultry campylobacters for epidemiological investigations

A technique for subtyping Camplobacter jejuni isolates has been developed by using the restriction fragment length polymorphism (Rnp) of polymerase chain reaction (PCR) products of the fluA and flaB genes. The technique was validated by using strains representing 28 serotypes of C jejuni and it may also be applied to C coli. From these strains 12 distinct RFLP profiles were observed but there was no direct relationship between the RFLP profile and the serotype. One hundred and thirty-five campylobacter isolates from 15 geographically distinct broiler flocks were investigated. All the isolates could be subtyped by using the RFLP method. Isolates from most of the flocks had a single RFLP profile despite data indicating that several serotypes were involved. Although it is possible that further restriction analysis may have demonstrated profile variations in these strains, it is more likely that antigenic variation can occur within genotypically related campylobacters. As a result, serotyping may give conflicting information for veterinary epidemiological purposes. This RFLP typing scheme appears to provide a suitable tool for the investigation of the sources and routes of transmission of campylobacters in chickens.

Prevalence of multiple antibiotic resistance in 443 Campylobacter spp. isolated from humans and animals

Aims: In view of recent findings that a multidrug efflux pump CmeABC exists in Campylobacter jejuni, 391 C. jejuni and 52 Campylobacter coli of human and animal origin were examined for a multidrug resistance phenotype. Materials and methods: The MICs of ampicillin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, tetracycline, cetrimide, triclosan, acridine orange, paraquat and ethidium bromide were determined. Resistance to organic solvents and the effect of salicylate (known inducer of the marRAB operon in Escherichia coli and Salmonella) were also examined. Results: Two C. coli and 13 C. jejuni isolates, mainly from pigs or poultry, were resistant to three or more antibiotics and 12 of these strains had reduced susceptibility to acridine orange and/or ethidium bromide. Strains (n=20) that were less susceptible to acridine orange, ethidium bromide and triclosan were significantly more resistant (P<0.05) to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, nalidixic acid and tetracycline, with two- to four-fold increases in MIC values compared with strains (n=20) most susceptible to acridine orange, ethidium bromide and triclosan. Growth of strains with 1 mM salicylate caused a small (up to two-fold) but statistically significant (Pless than or equal to0.005) increase in the MICs of chloramphenicol, ciprofloxacin, erythromycin and tetracycline. Conclusions: These data indicate that multiple antibiotic resistant (MAR)-like Campylobacter strains occur and it may be postulated that these may overexpress cmeABC or another efflux system.

Incidence of Campylobacter in pigs with and without diarrhea

Um total de 200 suínos (1 - 21 semanas de idade), originários de cinco criações localizadas no Estado de São Paulo, Brasil foi dividido em dois grupos de 100 animais caracterizando-se o grupo G1 de animais com diarréia e G2, sem diarréia. Campylobacter foi isolado em 43% das amostras provenientes de G1 e 34% de G2. O microrganismo foi mais frequentemente encontrado em leitões na faixa de 0 a 4 semanas de idade. Campylobacter coli foi a espécie mais comumente observada em G1 (44,2%) e em G2 (32,4%), seguido por Campylobacter jejuni/ coli com 16,3% em G1 e 23,5% em G2. As contagens de Campylobacter foram significativamente maiores (p < 0,01) em G1 ( £ 108 UFC/g) do que em G2 (£ 104 UFC/g), fato que sugere que o microrganismo pode, pelo menos, atuar no agravamento do processo diarréico.

CAMPYLOBACTER EM CAES COM E SEM DIARREIA: INCIDENCIA E SUSCETIBILIDADE A 21 ANTIMICROBIANOS

Two hundred and four stools of 102 diarrheic (0-12 months of age) and 102 non-diarrheic dogs coming from kennel and ambulatory, respectively, were assayed for the presence of Campylobacter. From the diarrhetic group, 46% of the samples yelded positive Campylobacter isolation mainly found in young animals until 5 months of age (72%), whereas those with age ranging from 6-12 months showed either a lower frequence of the organisms (28%) and a trend of the younger animals to be more charged than the older. 47 Campylobacter strains isolated from the diarrheic group were: C. jejuni biotype 1 (49%); C. jejuni biotyp 2 (11%); C. jejuni/coli (19%); C. coli (8,5%); Campylobacter NARTC group (8,5%) and C. sputoruns (4%). In the non-diarrhetic group, 27 (28%) Campylobacter strains were classified as: C. jejuni biotype 1 (34%) and biotype 2 (28%); C. jejuni/coli (24%) and C. coli (14%). According the biochemical tests, the 1% glycine tolerance test was not taking in account for the differentiation of C. jejuni because 45% of the strains failed in showing characteristic and 3 strains did not reduce the sodium selenite. The biochemical studies also showed phenotipical cross reactions between two Campylobacter NARTC-group strains with the C. jejuni strains, as well as two thermophilic species grew also at 25°C. All the 76 isolates were sensitive to gentamicin, nitrofurantoin and neomycin and resistant to oxacillin and penicillin. Furthermore, for the remaining 16 drugs the populational resistance ranged from 8% to 73% of strains. The presence of Campylobacter in dogs as well their close contact which man makes possible the occurrence of infections as also confirm the campylobacteriosis as an important zoonosis.

Freqüência de isolamento de Campylobacter em gatos com e sem diarréia e sensibilidade a antimicrobianos das estirpes isoladas

Pós-graduação em Medicina Veterinária - FMVZ

Aspectos microbiológicos e epidemiológicos da doença diarréica aguda no munic��pio de Juruti, Pará

O presente estudo descreveu os aspectos epidemiológicos e etiológicos da diarréia aguda no munic��pio de Juruti, Pará, Brasil. Foram avaliadas 261 amostras de fezes (170 diarréicas e 91 controles) de pacientes atendidos em Unidades de Saúde Pública, no período de fevereiro a julho de 2009. Para o isolamento de bactérias enteropatogênicas, utilizou-se meios seletivos indicadores e de enriquecimento. A caracterização bioquímica foi realizada utilizando os Sistemas API-20E e a sorologia, através de antisoros polivalentes e monovalentes. Para a detec��ão das categorias de E. coli diarreiogênicas foram executados dois ensaios de PCR multiplex. A pesquisa de Campylobacter jejuni e Campylobacter coli e Rotavírus foi executada através da técnica de ELISA, nas amostras de fezes. No exame parasitológico foram utilizados os métodos diretos (salina/Lugol) e sedimentação espontânea. Das 154 amostras positivas (118 diarréicas e 36 controles), 75,4% eram de infec��ões únicas e 24,6% de infec��ões mistas. A maioria dos casos incluiu crianças menores de 10 anos de idade (55,9%), sem diferença significante entre os sexos feminino e masculino. Os enteropatógenos mais frequentes no grupo diarréico foram E. histolytica/E. dispar (26%), Shigella spp (15,7%), G. lamblia (13,3%) e E. coli diarreiogênicas (12,8%), com Shigella spp associada à diarréia aguda (p = 0,0028). Quanto às categorias patogênicas de E. coli, a ETEC (7,2%) foi o tipo mais frequente nos casos de diarréia aguda, seguido de EAEC (5,9%). Os agentes menos frequentes nos casos diarréicos foram representados por C. jejuni/C. coli (4,7%), Rotavírus (2,8%), Salmonella Panama, A. hydrophila e A. sóbria (0,5%). Os resultados encontrados fornecem subsídios importantes para a vigilância epidemiológica e ambiental da doença diarréica aguda, no munic��pio de Juruti.

Pesquisa de Campylobacter spp. em granjas e abatedouro avícolas na mesorregião metropolitana de Belém - PA

As infec��ões de origem alimentar no homem, causadas por Campylobacter spp., resultam em grandes perdas econômicas e estão relacionadas à produção e o abate de frangos, etapas importantes na disseminação dessas bactérias. Baseando-se na importância do Campylobacter spp. na saúde pública e tendo em vista os dados constantes na literatura de que as aves comercializadas estão constantemente contaminadas com esse agente, sentiu-se a necessidade de realizar um estudo envolvendo a criação e o abate de frangos na região amazônica para que medidas profiláticas e de controle possam ser adotadas. O trabalho teve como objetivo estudar a ocorrência de Campylobacter spp. em granjas e abatedouro avícolas na mesorregião metropolitana de Belém – PA; isolar e identificar as espécies de Campylobacter spp. e identificar as fontes de contaminação nas granjas e os pontos críticos no abate. Foi coletado um total de 120 amostras em três granjas avícolas: 30 amostras de “swab” cloacal, 30 amostras de cama de frango, 30 amostras de ração e 30 amostras de água dos bebedouros. No abatedouro, foram colhidas 126 amostras: 36 amostras de água em 12 pontos diferentes da linha de abate e mais 30 amostras de pele do conjunto peito/ pescoço, 30 amostras de fígado e 30 amostras de moela. As amostras foram colhidas entre os meses de janeiro e maio de 2007 para o isolamento e identificação das espécies de Campylobacter spp. As amostras foram processadas na Seção de Bacteriologia e Micologia do Instituto Evandro Chagas – IEC da Secretaria de Vigilância em Saúde (SVS), Ministério da Saúde, Ananindeua – PA. Campylobacter spp. foi isolado em 33,3% (40/120) das amostras das granjas. Não houve diferença significativa (p>0,05) entre os percentuais de isolamentos positivos entre as três granjas pesquisadas. Ao analisar as freqüências dos isolados de Campylobacter spp. para cada tipo de amostra das granjas, observou-se que 96,6% (29/30) das amostras de “swab” cloacal, 33,3% (10/30) das amostras de cama e 3,3% (1/30) das amostras de água foram positivas para Campylobacter spp. Não foi isolada a bactéria nas amostras de ração. Campylobacter jejuni foi identificado bioquimicamente em 82,5% (33/40) das cepas isoladas nas granjas. No abatedouro, todas as cepas isoladas foram identificadas como C. jejuni., sendo isolado a bactéria em 8,73% (11/126) das amostras provenientes da linha de abate. Ao analisar as freqüências dos isolados de C. jejuni para cada tipo de amostra do abatedouro, observou-se que 27,8% (10/36) das amostras de água foram positivas para C. jejuni, seguido pela moela com 3,3% (1/30) das amostras positivas. Não foi isolado Campylobacter spp. nas amostras de fígado e de pele do conjunto peito/ pescoço. Houve diferença significativa (p<0,0001) entre os isolamentos positivos, negativos e os tipos de amostras processadas nas granjas e no abatedouro. As principais fontes de contaminação nas granjas foram o “swab” cloacal, a cama e, em menor escala, a água. Os principais pontos críticos observados no abatedouro foram a água, seguido pela moela. C. jejuni foi identificado em elevado percentual entre as cepas isoladas nas granjas e em todas as cepas do abatedouro.

Monospecific high-affinity and complement activating anti-GM1 antibodies are determinants in experimental axonal neuropathy

It has been difficult to replicate consistently the experimental model of axonal Guillain-Barré syndrome (GBS). We immunized rabbits with two lipo-oligosaccharides (LOS1 and LOS2) derived from the same C. jejuni strain and purified in a slightly different way. LOS1 did not contain proteins whereas several proteins were present in LOS2. In spite of a robust anti-GM1 antibody response in all animals the neuropathy developed only in rabbits immunized with LOS1. To explain this discrepancy we investigated fine specificity, affinity and ability to activate the complement of anti-GM1 antibodies. Only rabbits immunized with LOS1 showed monospecific high-affinity antibodies which activated more effectively the complement. Although it is not well understood how monospecific high-affinity antibodies are induced these are crucial for the induction of experimental axonal neuropathy. Only a strict adherence to the protocols demonstrated to be successful may guarantee the reproducibility and increase the confidence in the animal model as a reliable tool for the study of the human axonal GBS.

Genotype and antibiotic resistance analyses of Campylobacter isolates from ceca and carcasses of slaughtered broiler flocks

To obtain genetic information about Campylobacter jejuni and Campylobacter coli from broilers and carcasses at slaughterhouses, we analyzed and compared 340 isolates that were collected in 2008 from the cecum right after slaughter or from the neck skin after processing. We performed rpoB sequence-based identification, multilocus sequence typing (MLST), and flaB sequence-based typing; we additionally analyzed mutations within the 23S rRNA and gyrA genes that confer resistance to macrolide and quinolone antibiotics, respectively. The rpoB-based identification resulted in a distribution of 72.0% C. jejuni and 28.0% C. coli. The MLST analysis revealed that there were 59 known sequence types (STs) and 6 newly defined STs. Most of the STs were grouped into 4 clonal complexes (CC) that are typical for poultry (CC21, CC45, CC257, and CC828), and these represented 61.8% of all of the investigated isolates. The analysis of 95 isolates from the cecum and from the corresponding carcass neck skin covered 44 different STs, and 54.7% of the pairs had matching genotypes. The data indicate that cross-contamination from various sources during slaughter may occur, although the majority of Campylobacter contamination on carcasses appeared to originate from the slaughtered flock itself. Mutations in the 23S rRNA gene were found in 3.1% of C. coli isolates, although no mutations were found in C. jejuni isolates. Mutations in the gyrA gene were observed in 18.9% of C. jejuni and 26.8% of C. coli isolates, which included two C. coli strains that carried mutations conferring resistance to both classes of antibiotics. A relationship between specific genotypes and antibiotic resistance/susceptibility was observed.

Source attribution of human Campylobacter isolates by MLST and fla-typing and association of genotypes with quinolone resistance

Campylobacteriosis is the most frequent zoonosis in developed countries and various domestic animals can function as reservoir for the main pathogens Campylobacter jejuni and Campylobacter coli. In the present study we compared population structures of 730 C. jejuni and C. coli from human cases, 610 chicken, 159 dog, 360 pig and 23 cattle isolates collected between 2001 and 2012 in Switzerland. All isolates had been typed with multi locus sequence typing (MLST) and flaB-typing and their genotypic resistance to quinolones was determined. We used complementary approaches by testing for differences between isolates from different hosts with the proportion similarity as well as the fixation index and by attributing the source of the human isolates with Bayesian assignment using the software STRUCTURE. Analyses were done with MLST and flaB data in parallel and both typing methods were tested for associations of genotypes with quinolone resistance. Results obtained with MLST and flaB data corresponded remarkably well, both indicating chickens as the main source for human infection for both Campylobacter species. Based on MLST, 70.9% of the human cases were attributed to chickens, 19.3% to cattle, 8.6% to dogs and 1.2% to pigs. Furthermore we found a host independent association between sequence type (ST) and quinolone resistance. The most notable were ST-45, all isolates of which were susceptible, while for ST-464 all were resistant.

Genotypes and antibiotic resistance of bovine Campylobacter and their contribution to human campylobacteriosis

Campylobacter jejuni and Campylobacter coli are the most important bacterial causes of human gastroenteritis. Chicken has been recognized as a major source for human infection, whereas cattle might also contribute to a lesser extent. However, there is a paucity of information available regarding Campylobacter in Swiss cattle and their role for human campylobacteriosis. To gain more information on genotypes and antibiotic resistance of bovine C. jejuni and C. coli and on their contribution to human disease, 97 cattle isolates were analysed. Multilocus sequence typing (MLST) and flaB typing were applied and the gyrA and 23S rRNA genes were screened for point mutations responsible for quinolone and macrolide resistance, respectively. A total of 37 sequence types (STs) and 44 flaB types were identified, including two sequence types and five flaB types not previously described. Most common sequence types were ST21 (21%), ST61 (12%) and ST48 (11%). Only one isolate was macrolide resistant while 31% (n = 30) were quinolone resistant. Source attribution indicated chicken as the main source of human infection with cattle being second. In conclusion, cattle should not be underestimated as a potential source of human campylobacteriosis.

Novel Anti-Campylobacter Compounds Identified Using High Throughput Screening of a Pre-selected Enriched Small Molecules Library

Campylobacter is a leading cause of foodborne bacterial gastroenteritis worldwide and infections can be fatal. The emergence of antibiotic-resistant Campylobacter spp. necessitates the development of new antimicrobials. We identified novel anti-Campylobacter small molecule inhibitors using a high throughput growth inhibition assay. To expedite screening, we made use of a “bioactive” library of 4,182 compounds that we have previously shown to be active against diverse microbes. Screening for growth inhibition of Campylobacter jejuni, identified 781 compounds that were either bactericidal or bacteriostatic at a concentration of 200 µM. Seventy nine of the bactericidal compounds were prioritized for secondary screening based on their physico-chemical properties. Based on the minimum inhibitory concentration against a diverse range of C. jejuni and a lack of effect on gut microbes, we selected 12 compounds. No resistance was observed to any of these 12 lead compounds when C. jejuni was cultured with lethal or sub-lethal concentrations suggesting that C. jejuni is less likely to develop resistance to these compounds. Top 12 compounds also possessed low cytotoxicity to human intestinal epithelial cells (Caco-2 cells) and no hemolytic activity against sheep red blood cells. Next, these 12 compounds were evaluated for ability to clear C. jejuni in vitro. A total of 10 compounds had an anti-C. jejuni effect in Caco-2 cells with some effective even at 25 µM concentrations. These novel 12 compounds belong to five established antimicrobial chemical classes; piperazines, aryl amines, piperidines, sulfonamide and pyridazinone. Exploitation of analogues of these chemical classes may provide Campylobacter specific drugs that can be applied in both human and animal medicine.