Ömer Akineden

Enterobacteriaceae in dehydrated powdered infant formula manufactured in Indonesia and Malaysia

To determine the occurrence of Salmonella and Shigella in infant formula from Southeast Asia, 74 packages of dehydrated powdered infant follow-on formula (recommended age, > 4 months) from five different manufacturers, four from Indonesia and one from Malaysia, were analyzed. None of the 25-g test portions yielded Salmonella or Shigella. However, further identification of colonies growing on selective media used for Salmonella and Shigella detection revealed the frequent occurrence of several other Enterobacteriaceae species. A total of 35 samples (47%) were positive for Enterobacteriaceae. Ten samples (13.5%) from two Indonesian manufacturers yielded Enterobacter sakazakii. Other Enterobacteriaceae isolated included Pantoea spp. (n = 12), Escherichia hermanii (n = 10), Enterobacter cloacae (n = 8), Klebsiella pneumoniae subsp. pneumoniae (n = 3), Citrobacter spp. (n = 2), Serratia spp. (n = 2), and Escherichia coli (n = 2). To our knowledge, this is the first report to describe the contamination of dehydrated powdered infant formula from Indonesia with E. sakazakii and several other Enterobacteriaceae that could be opportunistic pathogens. Improper preparation and conservation of these products could result in a health risk for infants in Indonesia.

Identification of Streptococcus canis Isolated from Milk of Dairy Cows with Subclinical Mastitis

ABSTRACT Streptococcus canis was isolated from 31 milk samples from 11 cows in a dairy herd (with 49 lactating cows) affected by subclinical mastitis in north Rhine-Westphalia, Germany. Thirty-one isolates from the infected udder quarters were further characterized for their phenotypic and molecular properties. Most isolates (83.9%) produced α-galactosidase, and all were negative for β-d-glucuronidase. Amplification of the 16S rRNA gene by the PCR method and digestion with the restriction enzymes RsaI, MspI, and AvaII yielded species-specific patterns. Additional identification by species-specific amplification of the 16S rRNA gene, the 16S-23S rRNA gene intergenic spacer region, the CAMP factor-encoding gene cfg, and the internal fragments of the sodA gene was consistent with S. canis. Macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis showed that the S. canis isolates originated from a single clone or were very closely related.

Genetic relatedness of methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolated from a dog and the dog owner.

In the present study four methicillin-resistant Staphylococcus pseudintermedius (MRSP) strains isolated from a dog (n=3) and the anterior nares of the dog owner (n=1) were investigated by conventional and molecular methods. The species identity of the four S. pseudintermedius strains was confirmed by conventional methods, by PCR mediated amplification of S. intermedius/S. pseudintermedius specific segments of thermonuclease encoding gene nuc and by restriction fragment length polymorphism analysis of phosphoacetyltransferase encoding gene pta. Investigation of the four S. pseudintermedius for toxinogenic potential revealed that all four strains were positive for the exfoliative toxin encoding gene siet and the leukotoxin encoding genes lukS, lukF. The oxacillin and penicillin resistance of the four S. pseudintermedius strains could be determined by cultivation of the strains on oxacillin resistant screening agar base, ChromID MRSA Agar and Brilliance MRSA Agar and by multiplex PCR detecting the resistance genes mecA and blaZ. The genetic relatedness of the strains was studied by macrorestriction analysis of their chromosomal DNA using pulsed field gel electrophoresis (PFGE). According to PFGE all four S. pseudintermedius strains represent an identical bacterial clone indicating a cross transmission between the dog and the dog owner.

Presence of intestinal Mycobacterium avium subspecies paratuberculosis(MAP) DNA is not associated with altered MMP expression in ulcerative colitis

BackgroundMycobacterium avium subspecies paratuberculosis (MAP) is suspected to be a causative agent in human Crohns disease (CD). Recent evidence suggests that pathogenic mycobacteria and MAP can induce the expression of Matrix Metalloproteinases (MMP), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within this study we assessed the prevalence of intestinal MAP specific DNA in patients with Crohns disease, ulcerative colitis (UC), and healthy controls. We further analysed regulation patterns of MMPs in mucosal tissues of UC patients with and without intestinal MAP DNA detection.MethodsColonic biopsy samples were obtained from 63 Norwegian and German IBD patients and 21 healthy controls. RNA was quantified by quantitative real-time polymerase chain reaction (PCR) to study MMP gene expression in both pathological and healthy mucosal specimens. The presence of MAP DNA in colonic mucosa was examined using MAP specific PCR.ResultsMAP DNA was detected in 20% of UC patients and 33% of healthy controls but only in 7% of patients with CD. UC patients treated with corticosteroids exhibited a significantly increased frequency of intestinal MAP DNA compared to those not receiving corticosteroids. Expression of MMP-1, -2, -7, -9, -13, -19, -28 and TNF-α did not differ between UC patients with presence of intestinal MAP DNA compared to those without. MMP-2, MMP-9 and MMP-13 were significantly decreased in UC patients receiving corticosteroids.ConclusionsThe presence of intestinal MAP specific DNA is not associated with altered MMP expression in UC in vivo. Corticosteroids are associated with increased detection of intestinal MAP DNA and decreased expression of certain MMPs. Frequent detection of MAP DNA in healthy controls might be attributable to the wide environmental distribution of MAP and its presence in the food-chain.

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis at a regional scale in Germany.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of Johnes disease in dairy cattle. Genotyping of MAP is useful to gain a better understanding of the origin of infection, to evaluate regional control programs, to improve diagnostics, and to develop vaccines. In this study 91 MAP isolates mainly from symptomatic dairy cattle in Rhineland-Palatinate (RP, Germany), its neighbor federal states, and Luxembourg were genotyped using Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeat (MIRU-VNTR) and Multilocus Short Sequence Repeats (MLSSR). MIRU-VNTR and MLSSR produced 11 and 6 different genotypes among the 91 isolates, respectively. The combined analysis of both methods produced 25 genotypes with an index of discrimination (D) of 0.93 (95% CI: 0.91-0.95). The results revealed the genetic diversity of MAP and the dominance of two MAP genotypes commonly found in Europe, showed the usefulness of MAP genotyping in studies at a regional scale, and provided useful information for control initiatives in RP.

Extended-spectrum β-lactamase producing Enterobacteriaceae in bulk tank milk from German dairy farms.

Although the dairy farm environment is a known source of extended-spectrum β-lactamase (ESBL)-producing bacteria, surveillance data on ESBL in the milk production chain are still scarce. This study aimed at estimating the dimensions of the problem for public health and animal welfare by surveying ESBL-producing Enterobacteriaceae in raw bulk tank milk in Germany. Samples from 866 dairy farms, comprising about 1% of the total number of dairy farms in Germany, were first screened for presence of cefotaxime-resistant bacteria by selective enrichment. Suspect colonies were identified phenotypically and further characterized by biochemical and molecular methods, including analysis of resistance genes and clonal diversity in ESBL-producing isolates. Bulk tank milk from 82 (9.5%) farms yielded Enterobacteriaceae with confirmed ESBL-production. The most frequent ESBL-producing species was Escherichia coli (75.6%), followed by Citrobacter spp. (9.6%), Enterobacter cloacae (6.1%), and Klebsiella oxytoca (3.7%), a few isolates belonged to other species within the genera Hafnia, Raoutella and Serratia. The majority of isolates (95.1%) harbored the β-lactamase blaCTX-M gene, which has gained increased importance among ESBL-producing strains worldwide; the CTX-M group 1 was found to be the dominating (88.4%) phylogenetic group. All ESBL-positive Escherichia coli isolates were clonally heterogeneous, as determined by pulsed-field gel electrophoresis. The results from this survey demonstrate that ESBL-producing bacteria are distributed widely in the dairy farm environment in Germany. Therefore, raw milk is a potential source of exposure for the consumer, which is of increasing importance considering the trend of farmer-to-consumer direct marketing. Furthermore, dairy farm staff have an increased likelihood of exposure to ESBL-producing bacteria. Finally, ESBL-producing bacteria may also be transferred via waste milk to calves, thus further spreading antibiotic resistance in the farm environment.

Induction of matrix metalloproteinases and TLR2 and 6 in murine colon after oral exposure to Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP) is suspected to be a causative agent in Crohns disease. Recent evidence suggests that MAP can induce the expression of Matrix Metalloproteinases (MMPs), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within the present study, we analysed whether oral MAP exposure can induce colonic MMP expression in vivo. In MAP exposed mice MAP and spheroplasts were visualized in intramucosal leukocyte aggregates. MAP exposed mice exhibited a higher colonic expression of Mmp-2, -9, -13, -14, Timp-1, Tlr2, Tlr6, Il-1β, and Tnf-α. Cell clusters of MMP-9 positive cells adjacent to intramucosal leukocyte aggregates and CD45(+) leukocytes were identified as the major cellular sources of MMP-9. Enhanced TLR2 expression was visualized on the luminal side of colonic enterocytes. Although MAP exposure did not lead to macroscopic intestinal inflammation, the observed MAP spheroplasts in intramucosal leukocyte aggregates together with increased colonic expression of toll-like receptors, pro-inflammatory cytokines, and MMPs upon MAP exposure represents a part of the host immune response towards MAP.

Microbiological Quality of Raw Dried Pasta from the German Market, with Special Emphasis on Cronobacter Species.

The microbiological quality of 132 dried pasta products available on the German market, originating from 11 different countries, was studied. Sample materials included soft or durum wheat products, some of which produced with other ingredients such as eggs, spices, or vegetables. Parameters included hygiene indicators (aerobic plate count, mold count, the presence of Enterobacteriaceae) and pathogenic/toxinogenic bacterial species (Salmonella spp., Staphylococcus aureus, presumptive Bacillus cereus, and Cronobacter spp.). The overall results of hygiene parameters indicated a satisfactory quality. Salmonella was not found in any sample. Three samples were positive for S. aureus (10(2) to 10(4) colony forming unit (CFU)/g). Presumptive B. cereus at levels of 10(3) to 10(4) CFU/g were detected in 3 samples. Cronobacter spp. were isolated from 14 (10.6%) products. Of these, 9 isolates were identified as C. sakazakii, 2 each as C. turicensis and C. malonaticus, and 1 as C. muytjensii. The isolates were assigned to 9 multilocus sequence typing (MLST) sequence types and to 14 different PFGE profiles. Although pasta products are typically cooked before consumption, some consumers, and children in particular, may also eat raw pasta as nibbles. Raw pasta seems to be a relevant source of exposure to dietary Cronobacter spp., although health risks are probably restricted to vulnerable consumers. High numbers of presumptive B. cereus as found in some samples may be a risk after improper storage of cooked pasta products because toxinogenic strains are frequently found within this species.

Serological and molecular detection of Mycobacterium avium subsp. paratuberculosis in cattle of dairy herds in Colombia

The objective of this study is the detection of Mycobacterium avium subsp. paratuberculosis (MAP) by serum enzyme-linked immunosorbent assay (ELISA), fecal polymerase chain reaction (PCR), and fecal culture in Colombian dairy herds. Serum and fecal samples from asymptomatic cows (n = 307) of 14 dairy herds were tested for MAP by an unabsorbed ELISA test (ELISA-A). Serum and fecal samples from positive ELISA-A animals (n = 31) were further tested by an absorbed ELISA test (ELISA-B) and PCR. Fecal samples from animals of herds positive by ELISA-A and PCR (n = 105) were inoculated onto three different culture media. ELISA-A produced positive results in 10% of the serum samples and 71% of the herds. ELISA-B and PCR results were positive in two and six serum and fecal samples from positive ELISA-A animals, respectively. Fecal samples were negative for MAP on all culture media. The results of this study confirmed the presence of MAP in local dairy herds and the difficulties of MAP detection in asymptomatic animals by ELISA, PCR, and fecal culture.