Clonal spread of non-O157 Shiga toxigenic Escherichia coli O21:H25 in raw water buffalo milks.

AIMS: This study was conducted to investigate the presence of Shiga toxin-producing O157 and non-O157 E. coli in raw water buffalo milk, as well as to determine the virulence gene profiles, phylogroups, sequence types, and serotypes of the isolated strains. METHODS AND RESULTS: A total of 200 hand-milked raw water buffalo milk samples were collected from 200 different water buffaloes over a period of three months from 20 different farms. Isolation of STEC was performed using CHROMagar STEC. Presence of stx1, stx2, and eaeA genes were investigated by mPCR. Phylogroups and sequence types of E. coli strains were determined by Clermont phylotyping and MLST. Serotyping was performed using PCR or WGS. According to the results, two milk samples obtained from two different farms were found as STEC-positive. All Stx-positive E. coli isolates belonged to phylogenetic group A and were assigned to ST10. WGS results indicated that serotype of two isolates was O21:H25 and average nucleotide identity was detected at 99.99%. Thirteen additional registered E. coli O21:H25 assembled WGS data were obtained from EnteroBase and a phylogenetic tree was constructed. CONCLUSIONS: With this study, the presence of stx2 harboring E. coli O21:H25 in milk was identified for the first time. Although the identified serotype is considered a non-pathogen seropathotype, we conclude it could play an important role in the environmental circulation of Stx-phages and consequently contribute to the emergence of new STEC-related outbreaks.

Examining the presence of carbapenem resistant Enterobacterales and routes of transmission to bovine carcasses at slaughterhouses.

This study was conducted to investigate the existence and possible transmission routes of CREs during the bovine slaughter process. A total of 600 samples including rectoanal mucosal swaps, bovine hides and carcasses were collected weekly, over a 20 week period from three different slaughterhouses in Samsun province and analyzed in terms of CRE. Isolation of CRE was performed using Chromatic CRE Agar. Obtained isolates were identified using PCR and VITEK MS. E-test method was used for screening of carbapenemase production and disk diffusion method was used for detection of phenotypic carbapenem resistance. Presence of five major carbapenemase genes were investigated by PCR and obtained amplicons were sequenced by Sanger sequencing. Clonal relatedness was investigated by Clermont phylo-typing and MLST. Plasmid incompability groups were determined by PCR-based replicon typing. Based on the results, only one bovine hide sample was found positive in terms of CRE and blaKPC-2 harbouring E. coli ST398 (phylogroup A) was identified. E. coli ST398 was found resistant to meropenem, imipenem, ertapenem, doripenem and also tested fluoroquinolones. ST398 was found to harbour three distinct replicons, namely N, FIIK, and FIB KQ. Inc. groups for these replicons were identified as IncN and IncFIIK. On the other hand, no concrete evidence has been obtained to suggest that CREs are spreading at the slaughterhouse level. Conclusively, conducting further studies in areas such as farms, pens, and feedlots is necessary to gain a better understanding of the transmission routes of CREs in livestock.

Serotyping and antibiotic resistance of Listeria monocytogenes isolated from raw water buffalo milk and milk products.

This study was aimed to investigate the presence of Listeria monocytogenes in raw water buffalo milk and milk products, besides determining its serotype and the extent of its resistance against various antibiotics. A total of 188 samples of raw water buffalo milk and milk products were collected from Samsun Province, Turkey between November 2012 and May 2013. The classical culture technique was used to isolate and identify L. monocytogenes, as described in EN ISO 11290-1. The isolates were confirmed as L. monocytogenes by using PCR with (hylA) primers specific for the hemolysin gene. The antimicrobial susceptibility test was achieved by using the VITEK 2 compact system and VITEK 2 AST-P640 card. L. monocytogenes was found in 7 (3.7%) of the 188 samples. Four of them were obtained from cheese and three from milk samples. Whereas, L. monocytogenes was not detected in any of the clotted cream samples. A total of 13 isolates were confirmed by PCR as L. monocytogenes. Among these isolates, one was 1/2c (or 3c) (7.6%), three were 4b (or 4d, 4e) (23%), four were 1/2b (or 3b) (30.7%), and the other five isolates were serotype 1/2a (or 3a) (38.4%). The highest antimicrobial resistance was recorded against fosfomycine (100%) followed by oxacillin (92%), penicillin (84%), and erythromycin (69%). However, no resistance was determined against ciprofloxacin, gentamicin, and tigecycline. PRACTICAL APPLICATION: This study showed that some samples of raw buffalo milk and the milk products were contaminated with Listeria monocytogenes. The serotype with the highest prevalence was determined as L. monocytogenes 1/2a. This study also demonstrated that most of the L. monocytogenes isolates had developed multiresistance to many frequently used medical antimicrobial agents.

Detection of C. botulinum types in honey by mPCR.

The aim of this study was to determine the prevalence of Clostridium botulinum in honey samples using conventional methods and multiplex PCR (mPCR). A total number of 150 honey samples were randomly collected from apiaries, retail shops, weekly open bazaars, and supermarkets in Samsun, Turkey. Of 150 honey samples, 4 (2.6%) were positive for the botulinum neurotoxin gene by mPCR analysis. A total of 4 C. botulinum isolates were obtained from the mPCR positive samples, of which 3 were type A and 1 was type B. No samples were positive regarding the type E and type F neurotoxin genes. This is the first report of type A and type B spores of C. botulinum being detected and isolated in Turkey. This study revealed that some honey samples may present a potential hazard for food borne and infant botulism.

Determination of enterotoxigenic and methicillin resistant Staphylococcus aureus in ice cream.

The aim of this study was to determine the prevalence of enterotoxigenic and methicillin-resistant Staphylococcus aureus in ice creams. After culture-based identification of isolates, the presence of 16S rRNA and nuc was confirmed by mPCR. S. aureus was identified in 18 of 56 fruity (32.1%), 4 of 32 vanilla (12.5%), and 1 of 12 chocolate (8.3%) ice creams. S. aureus was identified as 38 isolates in 23 ice cream samples by culture-based techniques, but only 35 isolates were confirmed by PCR as S. aureus. To determine the enterotoxigenic properties of PCR-confirmed S. aureus isolates, a toxin detection kit was used (SET RPLA®). Of the 12 enterotoxigenic S. aureus isolates, 9 SEB (75%), 1 SED (8.3%), 1 SEB+SED (8.3%), and 1 SEA+SEB+SED (8.3%) expressing isolates were found. The presence of enterotoxin genes (sea, seb, sed) was identified in 13 (37.1%) out of 35 isolates by the mPCR technique. In the ice cream isolates, the sea, seb, and sed genes were detected: 1 sea (7.6%), 9 seb (69.2%), 1 sed (7.6%), 1 seb+sed (7.6%), and 1 sea+seb+sed (7.6%), respectively. The sec gene was not detected in any of these isolates. One of the 35 (2.8%) S. aureus strain was mecA positive.

Detection of enterotoxigenic Staphylococcus aureus in raw milk and dairy products by multiplex PCR.

The aim of this study was to investigate the prevalence of enterotoxigenic Staphylococcus aureus in 122 samples, including 60 raw milk, 32 white cheese, 10 kashar cheese, 10 butter, and 10 ice cream samples obtained from Samsun province, Turkey. In this study, S. aureus was detected in 64 samples, including raw milk (45/60; 75%), white cheese (12/32; 37.5%), kashar cheese (3/10; 30%), butter (3/10; 30%), and ice cream (1/10; 10%) samples. A total of 81 isolates were identified as S. aureus by PCR with the presence of 16S rRNA and nuc genes. The presence of genes encoding the staphylococcal enterotoxins (SEs) SEA, SEB, SEC, and SED was detected by multiplex PCR. According to the analysis, seven isolates from the raw milk samples (7/51; 13.7%) were enterotoxigenic; five of them produced SEA (5/7; 71.4%), one produced SEB (1/7; 14.2%), and one produced SEA+SEB (1/7; 14.2%). Four isolates from the white cheese samples (4/21; 19%) produced the SEA (1/4; 25%), SEC (1/4; 25%), SED (1/4; 25%), and SEA+SED (1/4; 25%) toxins. Two isolates from the kashar cheese samples (2/4; 50%) were found to be enterotoxigenic; one produced SEA (1/2; 50%) and the other produced SED (1/2; 50%). One isolate from the butter samples (1/4; 25%) showed enterotoxigenic character (SEB, 1/1; 100%). The products were found to be potentially hazardous to public health because of the fact that levels of contamination were higher than 105-106 cfu/g ml in 39% (25/64, 17 raw milk, 7 white cheese, and 1 butter) of the analyzed samples.

The prevalence of Escherichia coli O157 and O157:H7 in ground beef and raw meatball by immunomagnetic separation and the detection of virulence genes using multiplex PCR.

The present study was conducted to investigate the presence of Escherichia coli O157 and O157:H7 strains and to detect the presence of the stx1, stx2, and eaeA genes in isolates derived from 200 samples (100 samples from fresh ground beef and 100 samples from raw meatball). The samples were purchased from the Samsun Province in Turkey, over a period of 1 year. Enrichment-based immunomagnetic separation and multiplex polymerase chain reaction were applied for these analyses. E. coli O157 was detected in five of the 200 (2.5%) samples tested (one isolated from ground beef and four from meatball samples), whereas E. coli O157: H7 was not detected in any sample. During the analysis, eight strains of E. coli O157 were obtained. The genes stx1, stx2, and eaeA were detected in two E. coli O157 isolates obtained from two meatball samples, whereas only the eaeA and the stx2 genes were detected in four E. coli O157 strains that were isolated from one meatball sample. None of the stx1, stx2, and eaeA was detected in the E. coli O157 isolates obtained from the ground beef and the one meatball samples.