Implications of faecal indicator bacteria for the microbiological assessment of roof harvested rainwater quality in Southeast Queensland, Australia

The study aimed to evaluate the suitability of Escherichia coli, enterococci and C. perfringens to assess the microbiological quality of roof harvested rainwater, and to assess whether the concentrations of these faecal indicators can be used to predict the presence or absence of specific zoonotic bacterial or protozoan pathogens. From a total of 100 samples tested, respectively 58%, 83% and 46% of samples were found to be positive for E. coli, enterococci and C. perfringens spores, as determined by traditional culture based methods. Additionally, in the samples tested, 7%, 19%, 1%, 8%, 17%, and 15% were PCR positive for A. hydrophila lip, C. coli ceuE, C. jejuni mapA, L. pneumophila mip, Salmonella invA, and G. lamblia β-giardin genes. However, none of the samples was positive for E. coli O157 LPS, VT1, VT2 and C. parvum COWP genes. The presence or absence of these potential pathogens did not correlate with any of the faecal indicator bacterial concentrations as determined by a binary logistic regression model. The roof-harvested rainwater samples tested in this study appear to be of poor microbiological quality and no significant correlation was found between the concentration of faecal indicators and pathogenic microorganisms. The use of faecal indicator bacteria raises questions regarding their reliability in assessing the microbiological quality of water and particularly their poor correlation with pathogenic microorganisms. The presence of one or more zoonotic pathogens suggests that the microbiological analysis of water should be performed, and appropriate treatment measures should be undertaken especially in tanks where the water is used for drinking.

Campylobacter Infections of Domestic Origin in Finland

Campylobacter jejuni and C. coli are the leading causes of human bacterial gastroenteritis in developed countries. Most human Campylobacter infections are sporadic and a seasonal peak in the distribution of infections can be seen in the summer months in several countries, including Finland. A variety of risk factors for Campylobacter infections have been identified; handling and eating poultry, drinking unpasteurized milk, contact with domestic animals, and travelling abroad. However, the relative importance of the different risk factors in sporadic cases of Campylobacter infection remains unknown. In most cases, the infection is self-limiting and no specific treatment is required. Campylobacter enteritis can cause a wide range of complications, including reactive arthritis (ReA) that is reported in 1-5% of the cases. Seven clinical microbiology laboratories serving different geographical areas of Finland, participated in this multi-centre study, conducted during a seasonal peak in 2002. In a matched case-control study, domestically-acquired sporadic Campylobacter infections from three geographical areas were collected. The final study comprised 100 cases and 137 controls. Risk factors for sporadic domestically-acquired Campylobacter infections were identified on the basis of a questionnaire; swimming in natural waters was found to be a novel risk factor for Campylobacter infection. Other independent risk factors were tasting or eating raw or undercooked meat and drinking untreated water from a dug well. The role of bacterial strain and host characteristics are not fully understood in Campylobacter infections. Exposure factors, demographical characteristics, and the serotype of the Campylobacter isolate may affect the severity of the enteritis. This cross-sectional study comprised 114 patients with C. jejuni enteritis, diagnosed in three clinical microbiology laboratories; most of the patients had participated in the previous case-control study. Swimming was associated with age ≤ 5 years and serotype Pen 6,7 was found significantly more often among patients reporting swimming. The geographical distribution among serotypes varied; serotype Pen 4-complex appeared more often in patients from urban areas and serotype Pen 21 among patients from more rural areas. Thus, risk factors and sources of infection for C. jejuni infection may vary among individuals depending on age and geographical location. The in vitro susceptibilities of C. jejuni and C. coli strains isolated from patients infected abroad (85 strains) or domestically (393 strains) revealed that susceptibility to erythromycin is still high, even among isolates of foreign origin. However, the novel antimicrobial agent telithromycin did not offer any advantage over erythromycin; isolates with high minimal inhibitory concentrations (MICs) for erythromycin also showed reduced susceptibility to telithromycin. Reduced susceptibility to fluoroquinolones was detected almost exclusively among isolates of foreign origin and half of these isolates with high MICs for fluoroquinolones also showed elevated MICs for doxycycline. Questionnaires concerning complications associated with C. jejuni enteritis were sent to patients two months after becoming ill; 201 patients from seven different geographical areas were included in the study. Musculoskeletal complications after C. jejuni infection were commonly reported by patients (39%). The incidence of classical ReA was 4% and that of Achilles enthesopathy and/or heel pain 9%. Other C. jejuni-associated reactive joint symptoms were commonly reported, however, due to their milder nature seldom seen and diagnosed by a physician. The severity of the enteritis may predict further complications; stomach ache during enteritis was connected to the development of later joint pain. Early antimicrobial treatment, within two days from the start of symptoms, shortened the duration of diarrhoea by two days but did not prevent later musculoskeletal complications. Campylobacter is an important human enteropathogen and causes a significant burden of illness. As the incidence of Campylobacter infections is high, the importance of the infection and the occurrence of complications will increase. This stresses the importance of understanding the risk factors for acquiring Campylobacter infection and how bacterial strain and host characteristics may affect the risk for infection. The role of antimicrobial treatment for acute Campylobacter enteritis seems to be marginal and should be used restrictively.

Identification and Epidemiological Typing of Campylobacter strains isolated from Patients in Finland

C. jejuni constitutes the majority of Campylobacter strains isolated from patients in Finland, and C. coli strains are also reported. To improve the species identification, a combination of phenotype- and genotype-based methods was applied. Standardising the cell suspension turbidity in the hippurate hydrolysis test enabled the reliable identification of hippurate-positive Campylobacter strains as C. jejuni. The detection of species-specific genes by PCR showed that about 30% of the hippurate-negative strains were C. jejuni. Three typing methods, serotyping, PCR-RFLP analysis of LOS biosynthesis genes and pulsed-field gel electrophoresis (PFGE) were evaluated as epidemiological typing tools for C. jejuni. The high number of non-typeable strains lowered the discriminatory ability of serotyping. PCR-RFLP typing offered high discrimination for both serotypeable and non-typeable strains, but the correlation between serotypes and RFLP-types was not high enough to enable its use for molecular serotyping of non-typeable strains. PFGE was a highly discriminative typing method. Although the use of two restriction enzymes generally increases the discriminatory ability, KpnI alone offered almost as high discrimination as the use of SmaI and KpnI. The characteristic seasonal distribution of Campylobacter infections with a peak in summer and low incidence in winter was mainly due to domestically acquired infections. Of the C. jejuni strains, 41% were of domestic origin compared to only 17% of the C. coli strains. Serotypes Pen 12, Pen 6,7 and Pen 27 were significantly associated with domestic C. jejuni infections, Pen 1,44, Pen 3 and Pen 37 with travel-related infections. Pen 2 and Pen 4-complex were common both in domestic and travel-related infections. Serotype Pen 2 was less common among patients 60 years or older than in younger patients, more prevalent in Western Finland than in other parts of the country and more prevalent than other serotypes in winter. The source of Pen 2 infections may be related to cattle, since Pen 2 is the most common serotype in isolates from Finnish cattle. PFGE subtypes among isolates from patients and chickens during the summer 2003 and from cattle during the whole year were compared. The analysis of indistinguishable SmaI/KpnI subtypes suggested that up to 31% of the human infections may have been mediated by chickens and 19% by cattle. Human strains isolated during two one-year sampling periods were studied by PFGE. Of the domestic strains, 69% belonged to SmaI subtypes found during both sampling periods. Four SmaI subtypes accounted for 45% of the domestic strains, further typing of these subtypes by KpnI revealed six temporally persistent SmaI/KpnI subtypes. They were only occasionally identified in travel-related strains, and therefore, can be considered to be national subtypes. Each subtype was associated with a serotype: Pen 2, Pen 12, Pen 27, Pen 4-complex, Pen 41, and Pen 57. Five of these subtypes were identified in cattle (S5/K27, S7/K1, S7/K2, S7/K5 and S64/K19), and two in chickens (S7/K1 and S64/K19) with a temporal association with human infections in 2003. Cattle are more likely potential sources of these persistent subtypes, since long-term excretion of Campylobacter strains by cattle has been reported.

Kampylobakteerit Korkeasaaren eläintarhan eläimissä

Kampylobakteerit on tunnistettu vasta viimeisten 30 vuoden aikana merkittäviksi ihmisten tautien aiheuttajiksi. Termofiiliset kampylobakteerit Campylobacter jejuni ja C. coli ovat yleisin bakteeriperäisten suolistoinfektioden syy maailmanlaajuisesti. Pääasiallinen oire on ripuli. C. jejuni voi aiheuttaa myös jälkitauteja, kuten reaktiivista niveltulehdusta, nokkosihottumaa tai kyhmyruusua (erytema nodosum). Näitä kuitenkin ilmaantuu harvoin (<1 %). Tärkein jälkitauti on Guillain-Barrén syndrooma, joka on akuutti ääreishermoja demyelinisoiva sairaus. Kesällä 2008 Helsingin yliopistossa toteutettiin tutkimus, jonka tarkoituksena oli kartoittaa kampylobakteerien esiintymistä Korkeasaaren eläintarhassa ja tunnistaa löytyneet kampylobakteerit lajitasolle. Aineisto koostui 196 tarhaeläimen ulostenäytteestä, kahdeksasta Korkeasaaressa vapaana olleen valkoposkihanhen ulostenäytteestä sekä 12 ympäristönäytteestä (vesinäytteitä). Vesinäytteet olivat eläinten kahluualtaista tai häkeissä kulkevista puroista. Ulostenäytteet olivat peräisin kaikkiaan 71 eläinlajista. Tutkituista eläinlajeista 40 oli nisäkkäitä, 26 oli lintuja ja viisi oli matelijoita. Näytteistä eristettyjen isolaattien tulosten perusteella ulostenäytteistä 21 (10,7 %) oli positiivisia kampylobakteerien suhteen. Eläinlajeja tarkasteltaessa kampylobakteerien suhteen positiivisia oli 17 (23,9 %) eläinlajia. Näistä kymmenen oli lintuja, kuusi nisäkkäitä ja yksi matelija. Positiiviseksi C. jejunin suhteen osoittautui 18 ulostenäytettä. Yhdestä emun näytteestä löytyi C. jejunin lisäksi myös C. coli. Kahdessa näytteessä, rubiini-ibis ja kannuskilpikonna, kasvoi jokin muu kampylobakteeri kuin C. jejuni tai C. coli. Eläinlajeja tarkasteltaessa voidaan todeta, että 15 (21,1 %) todettiin positiivisiksi C. jejunin suhteen. Näistä kymmenen oli lintuja ja viisi nisäkkäitä. C. coli löytyi vain yhdestä (1,4 %) eläinlajista (linnusta) sekä jokin muu kampylobakteeri kuin C. jejuni tai C. coli löytyi kahdesta (2,8 %) eläinlajista (yksi lintu ja yksi matelija). Vain yksi ulostenäyte osoittautui positiiviseksi arkobakteerin suhteen. Kyseessä oli rubiini-ibiksen näyte. Samasta näytteestä löydettiin myös C. jejuni. Kahdesta valkoposkihanhien näytteestä löytyi sekä helikobakteeri että C. jejuni. Viisi ympäristönäytettä osoittautui positiiviseksi arkobakteerien suhteen. Kolme positiivista näytettä oli lintualtaista ja kaksi nisäkkäiden altaista. Rubiini-ibiksen altaasta löytyi arkobakteerin lisäksi myös C. jejuni. Lähes kaikissa vesinäytteissä (9/12) oli koliformisia bakteereja Colilert® testillä yli määritysrajan (>2419,6 bakteeria 100 ml:ssa näytettä). Suurimmasta osasta vesinäytteitä (8/12) saatiin tulokseksi myös yli menetelmän määritysrajan E. colia. Kaikenkaikkiaan tulosten perusteella voi päätellä, että vierailijoiden riski saada kampylobakterioosi Korkeasaaren eläintarhassa on suhteellisen pieni. Ainoana suurempana riskitekijänä voisi pitää valkoposkihanhien ulostetta. Sitä on maassa runsaasti ja sitä saattaa myös kulkeutua kenkien mukana jopa kotiin asti. Eläintarhan työntekijöiden tulee muistaa hyvä käsihygienia oltuaan kosketuksissa joko eläimien kanssa tai käytyään näiden tarhoissa. Työntekijät saattavat myös levittää eläintarhan eläinten ulostetta saappaissaan vierailijoidenkin kulkureiteille.

Factors affecting the pressure resistance of some Campylobacter species

Aims: To compare pressure resistance between strains of Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter fetus, and to investigate the effect of suspending medium on pressure resistance of sensitive and more resistant strains. Methods and Results: Six strains of C. jejuni and four each of C. coli, C. lari and C. fetus were pressure treated for 10 min at 200 and 300 MPa. Individual strains varied widely in pressure resistance but there were no significant differences between the species C. jejuni, C. coli and C. lari. Campylobacter fetus was significantly more pressure sensitive than the other three species. The pressure resistance of C. jejuni cultures reached a maximum at 16-18 h on entry into stationary phase then declined to a minimum at 75 h before increasing once more. Milk was more baroprotective than water, broth or chicken slurry but did not prevent inactivation even of a resistant strain at 400 MPa. Conclusions: Pressure resistance varies considerably between species of Campylobacter and among strains within a species, and survival after a pressure challenge will be markedly influenced by culture age and food matrix. Significance and Impact of the Study: Despite the strain variation in pressure resistance and protective effects of food, Campylobacter sp. do not present a particular problem for pressure processing.

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).

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 município de Juruti, Pará

O presente estudo descreveu os aspectos epidemiológicos e etiológicos da diarréia aguda no município 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 detecção 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 infecções únicas e 24,6% de infecções 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 município de Juruti.

Survey on the spirilar flora of Lagomorphs

Members of the genera Campylobacter and Helicobacter have been in the spotlight in recent decades because of their status as animals and/or humans pathogens, both confirmed and emerging, and because of their association with food-borne and zoonotic diseases. First observations of spiral shaped bacteria or Campylobacter-like organisms (CLO) date back to the end of the 19th century, however the lack of adequate isolation methods hampered further research. With the introduction of methods such as selective media and a filtration procedure during the 1970s led to a renewed interest in Campylobacter, especially as this enabled elucidation of their role in human hosts. On the other hand the classification and identification of these bacteria was troublesome, mainly because of the biochemical inertness and fastidious growth requirements. In 1991, the taxonomy of Campylobacter and related organisms was thoroughly revised, since this revision several new Campylobacter and Helicobacter species have been described. Moreover, thanks to the introduction of a polyphasic taxonomic practice, the classification of these novel species is well-founded. Indeed, a polyphasic approach was here followed for characterizing eight isolates obtained from rabbits epidemiologically not correlated and as a result a new Campylobacter species was proposed: Campylobacter cuniculorum (Chapter 1). Furthermore, there is a paucity of data regarding the occurrence of spiral shaped enteric flora in leporids. In order to define the prevalence both of this new species and other CLO in leporids (chapter 2), a total of 85 whole intestinal tracts of rabbits reared in 32 farms and 29 capture hares, epidemiologically not correlated, were collected just after evisceration at the slaughterhouse or during necroscopy. Examination and isolation methods were varied in order to increase the sensibility level of detection, and 100% of rabbit farms resulted positive for C. cuniculorum in high concentrations. Moreover, in 3.53% of the total rabbits examined, a Helicobacter species was detected. Nevertheless, all hares resulted negative both for Campylobacter or Helicobacter species. High prevalence of C. cuniculorum were found in rabbits, and in order to understand if this new species could play a pathological role, a study on some virulence determinants of C. cuniculorum was conducted (Chapter 3). Although this new species were able to adhere and invade, exert cytolethal distending toxin-like effects although at a low titre, a cdtB was not detected. There was no clear relationship between source of isolation or disease manifestation and possession of statistically significantly levels of particular virulence-associated factors although, cell adhesion and invasion occurred. Furthermore, antibiotic susceptibility was studied (chapter 4) in Campylobacter and in Escherichia coli strains, isolated from rabbits. It was possible to find acquired resistance of C. cuniculorum to enrofloxacin, ciprofloxacin and erytromycin. C. coli isolate was susceptible to all antimicrobial tested and moreover it is considered as a wild-type strain. Moreover, E. coli was found at low caecal concentration in rabbits and 30 phenotypes of antibiotic resistance were founded as well as the high rate of resistances to at least one antibiotic (98.1%). The majority of resistances were found from strains belonging to intensive farming system. In conclusion, in the course of the present study a new species isolated from rabbits was described, C. cuniculorum, and its high prevalence was established. Nevertheless, in hare samples no Campylobacter and Helicobacter species were detected. Some virulence determinants were further analyzed, however further studied are needed to understand the potential pathogenicity of this new species. On the other hand, antimicrobial susceptibility was monitored both in C. cuniculorum and indicator bacteria and acquired resistance was observed towards some antibiotics, indicating a possible role of rabbitries in the diffusion of antibiotic resistance. Further studies are necessary to describe and evaluate the eventual zoonotic role of Campylobacter cuniculorum.

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.