IncN plasmids carrying bla CTX-M-1 in Escherichia coli isolates on a dairy farm

The aim of the study was to compare the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli bovine isolates on a conventional dairy cattle farm with high consumption of parenteral and intramammary cephalosporins (farm A) and on an organic dairy farm with no cephalosporin use (farm B). ESBL-producing E. coli were isolated from rectal swabs and milk filters by selective cultivation on MacConkey agar with cefotaxime (2mg/l). ESBL genes were identified by polymerase chain reaction (PCR) and sequencing, and the genetic diversity of the isolates was determined by XbaI pulsed field gel electrophoresis (PFGE). Conjugative transfer, incompatibility group, and restriction fragment length polymorphism (RFLP) profiles of the ESBL-carrying plasmids were studied. Higher prevalence (39%, n(rectal samples in cows)=309) of CTX-M-1-producing E. coli isolates was found on farm A compared to farm B (<1%, n(rectal samples in cows)=154; 0%, n(rectal samples in calves)=46). Using PFGE, the isolates from farm A were divided into nine pulsotypes. In all ESBL-positive isolates, the bla(CTX-M-1) gene was carried on 40 kb IncN conjugative plasmids of three related HincII restriction profiles. Horizontal gene transfer through transmission of IncN plasmids harboring bla(CTX-M-1) as well as clonal dissemination of a particular clone seems to be involved in dissemination of CTX-M-1-producing E. coli isolates in cows on the farm using cephalosporins in treating bacterial infections. The study demonstrates a possible role of cephalosporin use in the widespread occurrence of CTX-M-1-producing E. coli on the conventional dairy cattle farm compared to the organic farm.     

Phylogenetic groups and cephalosporin resistance genes of Escherichia coli from diseased food-producing animals in Japan

A total of 318 Escherichia coli isolates obtained from different food-producing animals affected with colibacillosis between 2001 and 2006 were subjected to phylogenetic analysis: 72 bovine isolates, 89 poultry isolates and 157 porcine isolates. Overall, the phylogenetic group A was predominant in isolates from cattle (36/72, 50%) and pigs (101/157, 64.3%) whereas groups A (44/89, 49.4%) and D (40/89, 44.9%) were predominant in isolates from poultry. In addition, group B2 was not found among diseased food-producing animals except for a poultry isolate. Thus, the phylogenetic group distribution of E. coli from diseased animals was different by animal species. Among the 318 isolates, cefazolin resistance (minimum inhibitory concentrations: ≥32 μg/ml) was found in six bovine isolates, 29 poultry isolates and three porcine isolates. Of them, 11 isolates (nine from poultry and two from cattle) produced extended spectrum β-lactamase (ESBL). The two bovine isolates produced bla_CTX-M-2, while the nine poultry isolates produced bla_CTX-M-25 (4), bla_SHV-2 (3), bla_CTX-M-15 (1) and bla_CTX-M-2 (1). Thus, our results showed that several types of ESBL were identified and three types of β-lactamase (SHV-2, CTX-M-25 and CTX-M-15) were observed for the first time in E. coli from diseased animals in Japan.     

Serogroup distribution,diversity of exotoxin gene profiles,and phylogenetic grouping of CTX-M-1- producing uropathogenic Escherichia coli

The emergence of CTX-M-1 producing Uropathogenic Escherichia coli (UPEC) has become a serious challenge. In addition to antimicrobial resistance, a number of virulence factors have been shown. Therefore, this study was designed to determine the prevalence of O- serogroups, phylogenetic groups, exotoxin genes, and antimicrobial resistance properties of CTX-M-1- producing UPEC. A total of 248 UPEC isolates were collected. The antibiotic resistance was performed, and PCR was used to detect the bla_CTX-M1, exotoxins, serogroups and phylogroups of UPEC. Of 248 isolates, 95 (38.3%) harbored bla_CTX-M-1. Of them, serogroups O1 and O25 were predominant, accounting for 20% and 13.7%, respectively. The hlyA was the dominant exotoxin gene (32.6%), followed by sat (28.4%), vat (22.1%), cnf (13.7%), picU (8.4%), and cdt (2.1%). The hlyA gene was significantly associated with pyelonephritis (P = 0.003). Moreover, almost half of the isolates (45.4%) belonged to phylogenetic group B2. Most of exotoxin genes were present in significantly higher proportions in group B2 isolates except cdt gene (P < 0.05). All of the isolates were susceptible to imipenem, nitrofurantoin, and fosfomycin. The CTX-M-1-producing UPEC strains causing nosocomial infections are more likely to harbor certain exotoxin genes, raising the possibility that this increase in virulence genes may result in an increased risk of complicated UTI.     

食品动物源产CTX-M-14大肠杆菌传播分子机制的演变

从保存的2002-2009年分离的食品动物源大肠杆菌中,挑选16株bla_CTX-M-14阳性菌,用PCR方法检测超广谱β-内酰胺酶(ESBLs)编码基因、PMQR耐药基因及其他重要抗生素耐药基因(rmtB和floR);通过脉冲场凝胶电泳(PFGE)及种族进化关系分析16株细菌的亲缘关系;通过接合转移试验、复制子分型和bla_CTX-M-14上下游插入元件的检测,分析产CTX-M-14大肠杆菌的传播分子机制。PCR检测结果表明,16株食品动物源产CTX-M-14大肠杆菌大多属于系统发育组A组,其次为B1和D组,没有B2组;PFGE分型结果表明,同一时间内不同动物间存在产CTX-M-14共生型大肠杆菌克隆的扩散传播,但养殖场内CTX-M-14主要是随质粒或其他元件进行水平传播;质粒复制子分型结果表明,携带bla_CTX-M-14的质粒属于IncK(3/14)、 IncF(5/14)、 IncHI2(1/14)、IncFIB 和 IncF(1/14)、IncHI1和IncN(2/14)、 IncI1(2/14)等,且随着时间推移,复制子的种类呈增多趋势。2002-2007年的菌株bla_CTX-M基因的上下游均检测到ISEcp1和IS903;但2009年菌株除了部分在上下游都可以检测到ISEcp1和IS903外,还有的只检测到上游的ISEcp1或下游的IS903;2002-2009年的菌均未检测到ISCR1。16株产CTX-M-14大肠杆菌除了携带其他ESBLs编码基因,如bla_CTX-M79和bla_TEM-135外,还携带其他重要抗生素耐药基因,如oqxA、floR、aac(6')-1b-cr及rmtB,而且2002-2009年大肠杆菌携带耐药基因的种类和数量逐年增多;接合转移试验发现,2002-2005年的菌株,bla_CTX-M-14往往发生单独转移,而2009年分离菌bla_CTX-M-14往往和floR或rmtB位于同一质粒上发生共同转移。这说明养殖场使用氨基糖苷类或氟苯尼考等任何一种抗生素,都可以筛选出产CTX-M-14大肠杆菌并促进其扩散,所以动物养殖过程中要慎用这些抗生素。     

Surveillance of dairy production holdings supplying raw milk to the farmhouse cheese sector for Escherichia coli O157, O26 and O111

Clinically healthy domestic animals can harbour Escherichia coli O157 and other verocytotoxigenic E. coli (VTEC) strains in their faeces. Milk filters can be used to microbiologically monitor direct milk secretion and environmental contamination for these pathogens. The aim of this study was to establish baseline data on the prevalence and characteristics of VTEC organisms in lactating animals (bovine, ovine and caprine) supplying milk to the farmhouse cheese sector, with particular emphasis on serogroups O157, O111 and O26. Fifty-six bovine, 13 caprine and 5 ovine herds/flocks, the majority of which supplying milk for farmhouse cheese production, were surveyed from May 2004 to July 2005. Milk filters were analysed by immunomagnetic separation followed by PCR, on a serogroup-specific basis for E. coli O157, O26 and O111. Positive isolates were examined using a multiplex PCR protocol, for their potential to produce verocytotoxins (vt1/vt2), the haemolysin-encoding gene (hlyA) and the gene encoding attaching and effacement (eae). Five verocytotoxigenic and 22 non-virulent E. coli O157 isolates were detected. Seventeen E. coli O26 isolates were also detected, four of which were verocytotoxigenic, seven isolates contained the eae gene only and six isolates were devoid of any of the virulence factors. The VTEC O157 and O26 isolates contained the hlyA and eae genes along with the verocytotoxin genes. No E. coli O111 isolates were detected. Some of the herds were positive on more than one occasion and multiple E. coli serogroups were isolated from the same milk filter sample. Although all food products tested were VTEC negative, routine surveillance for such pathogens in raw milk/raw milk products is of public health importance. Herd-level surveillance along with subsequent risk management action may be a cost-effective component of risk reduction strategies for food production, drinking water supplies and the protection of public health.     

Antimicrobial resistance in Escherichia coli isolates from frugivorous (Eidolon helvum) and insectivorous (Nycteris hispida) bats in Southeast Nigeria,with detection of CTX-M-15 producing isolates

Thirty-five Escherichia coli isolates obtained from the liver, spleen and intestines of 180 frugivorous and insectivorous bats were investigated for antimicrobial resistance phenotypes/genotypes, prevalence of Extended-Spectrum beta-lactamase (ESBL) production, virulence gene detection and molecular typing. Eight (22.9 %) of the isolates were multidrug resistant (MDR). Two isolates were cefotaxime-resistant, ESBL-producers and harbored the bla_CTX-M-15 gene; they belonged to ST10184-D and ST2178-B1 lineages. tet(A) gene was detected in all tetracycline-resistant isolates while int1 (n = 8) and bla_TEM (n = 7) genes were also found. Thirty-three of the E. coli isolates were assigned to seven phylogenetic groups, with B1 (45.7 %) being predominant. Three isolates were enteropathogenic E. coli (EPEC) pathovars, containing the eae gene (with the variants gamma and iota), and lacking stx1/stx2 genes. Bats in Nigeria are possible reservoirs of potentially pathogenic MDR E. coli isolates which may be important in the ecology of antimicrobial resistance at the human-livestock-wildlife-environment interfaces. The study reinforces the importance of including wildlife in national antimicrobial resistance monitoring programmes.     

Colistin resistance and plasmid-mediated mcr genes in Escherichia coli and Salmonella isolated from pigs,pig carcass and pork in Thailand,Lao PDR and Cambodia border provinces

BackgroundColistin and carbapenem-resistant bacteria have emerged and become a serious public health concern, but their epidemiological data is still limited.ObjectivesThis study examined colistin and carbapenem resistance in Escherichia coli and Salmonella from pigs, pig carcasses, and pork in Thailand, Lao PDR, and Cambodia border provinces.MethodsThe phenotypic and genotypic resistance to colistin and meropenem was determined in E. coli and Salmonella obtained from pigs, pig carcasses, and pork (n = 1,619). A conjugative experiment was performed in all isolates carrying the mcr gene (s) (n = 68). The plasmid replicon type was determined in the isolates carrying a conjugative plasmid with mcr by PCR-based replicon typing (n = 7). The genetic relatedness of mcr-positive Salmonella (n = 11) was investigated by multi-locus sequence typing.ResultsColistin resistance was more common in E. coli (8%) than Salmonella (1%). The highest resistance rate was found in E. coli (17.8%) and Salmonella (1.7%) from Cambodia. Colistin-resistance genes, mcr-1, mcr-3, and mcr-5, were identified, of which mcr-1 and mcr-3 were predominant in E. coli (5.8%) and Salmonella (1.7%), respectively. The mcr-5 gene was observed in E. coli from pork in Cambodia. Two colistin-susceptible pig isolates from Thailand carried both mcr-1 and mcr-3. Seven E. coli and Salmonella isolates contained mcr-1 or mcr-3 associated with the IncF and IncI plasmids. The mcr-positive Salmonella from Thailand and Cambodia were categorized into two clusters with 94%–97% similarity. None of these clusters was meropenem resistant.ConclusionsColistin-resistant E. coli and Salmonella were distributed in pigs, pig carcasses, and pork in the border areas. Undivided-One Health collaboration is needed to address the issue.     

Antimicrobial resistance in faecal Escherichia coli isolates from farmed red deer and wild small mammals. Detection of a multiresistant E. coli producing extended-spectrum beta-lactamase

Eighty-nine Escherichia coli isolates recovered from faeces of red deer and small mammals, cohabiting the same area, were analyzed to determine the prevalence and mechanisms of antimicrobial resistance and molecular typing. Antimicrobial resistance was detected in 6.7% of isolates, with resistances to tetracycline and quinolones being the most common. An E. coli strain carrying bla_CTX-M-1 as well as other antibiotic resistant genes included in an unusual class 1 integron (Intl1-dfrA16–bla_PSE-1-aadA2-cmlA1-aadA1-qacH-IS440-sul3-orf1-mef(B)Δ-IS26) was isolated from a deer. The bla_CTX-M-1 gene was transferred by conjugation and transconjugants also acquired an IncN plasmid. This strain was typed as ST224, which seems to be well adapted to both clinical and environmental settings. The phylogenetic distribution of the 89 strains varied depending on the animal host. This work reveals low antimicrobial resistance levels among faecal E. coli from wild mammals, which reflects a lower selective pressure affecting these bacteria, compared to livestock. However, it is remarkable the detection of a multi-resistant ESBL-E. coli with an integron carrying clinically relevant antibiotic-resistance genes, which can contribute to the dissemination of resistance determinants among different ecosystems.     

Spread of extended-spectrum beta-lactamase producing Escherichia coli isolates in Swedish broilers mediated by an incI plasmid carrying blaCTX-M-1

Background

The already high and increasing occurrence of extended-spectrum beta-lactamases (ESBL) producing Escherichia coli in European broiler populations is of concern due to the fact that third and fourth generation cephalosporins are deemed critically important in human medicine. In Sweden 34% of the broilers carry ESBL/pAmpC producing E. coli in their gut, despite the absence of a known selection pressure such as antimicrobial usages. The aim of the current study was to characterise a selection of E. coli strains carrying the bla_CTX-M-1, to determine if the spread was due to a specific clone.

Findings

Ten isolates carrying bla_CTX-M-1 from Swedish broilers belonged to eight different multi-locus sequence types with three isolates belonging to ST155. The ST155 isolates were identical as assessed by PFGE. The bla_CTX-M-1 was in all isolates carried on a plasmid of replicon type incI, which also transferred resistance to tetracycline and sulfamethoxazole.

Conclusion

The occurrence of ESBL-producing E. coli in the Swedish broilers is not due to the emergence of a single clone, but rather the spread of a specific incI plasmid carrying bla_CTX-M-1.     

Molecular characterization of quinolone resistance mechanisms and extended-spectrum β-lactamase production in Escherichia coli isolated from dogs

The increasing prevalence of antimicrobial resistances is now a worldwide problem. Investigating the mechanisms by which pets harboring resistant strains may receive and/or transfer resistance determinants is essential to better understanding how owners and pets can interact safely. Here, we characterized the genetic determinants conferring resistance to β-lactams and quinolones in 38 multidrug-resistant Escherichia coli isolated from fecal samples of dogs, through PCR and sequencing. The most frequent genotype included the β-lactamase groups TEM (n = 5), and both TEM + CTX-M-1 (n = 5). Within the CTX-M group, we identified the genes CTX-M-32, CTX-M-1, CTX-M-15, CTX-M-55/79, CTX-M-14 and CTX-M-2/44. Thirty isolates resistant to ciprofloxacin presented two mutations in the gyrA gene and one or two mutations in the parC gene. A mutation in gyrA (reported here for the first time), due to a transversion and transition (TCG → GTG) originating a substitution of a serine by a valine in position 83 was also detected. The plasmid-encoded quinolone resistance gene, qnrs1, was detected in three isolates. Dogs can be a reservoir of genetic determinants conferring antimicrobial resistance and thus may play an important role in the spread of antimicrobial resistance to humans and other co-habitant animals.     

Dissemination of pHK01-like incompatibility group IncFII plasmids encoding CTX-M-14 in Escherichia coli from human and animal sources

Few studies have compared CTX-M encoding plasmids identified in different ecological sources. This study aimed to analyze and compare the molecular epidemiology of plasmids encoding CTX-M-14 among strains from humans and animals. The CTX-M-14 encoding plasmids in 160 Escherichia coli isolates from animal faecal (14 pigs, 16 chickens, 12 cats, 8 cattle, 5 dogs and 3 rodents), human faecal (45 adults and 20 children) and human urine (37 adults) sources in 2002-2010 were characterized by molecular methods. The replicon types of the CTX-M-14 encoding plasmids were IncFII (n=61), I1-Iγ (n=24), other F types (n=23), B/O (n=10), K (n=6), N (n=3), A/C (n=1), HI1 (n=1), HI2 (n=1) and nontypeable (n=30). The genetic environment, ISEcp1 -bla(CTX-M-14) - IS903 was found in 89.7% (52/58), 87.7% (57/65) and 86.5% (32/37) of the animal faecal, human faecal and human urine isolates, respectively. Subtyping of the 61 IncFII incompatibility group plasmids by replicon sequence typing, plasmid PCR-restriction fragment length polymorphism and marker genes (yac, malB, eitA/eitC and parB/A) profiles showed that 31% (18/58), 30.6% (20/65) and 37.8% (14/37) of the plasmids originating from animal faecal, human faecal and human urine isolates, respectively, were pHK01-like. These 52 pHK01-like plasmids originated from diverse human (20 faecal isolates from 2002, 2007 to 2008, 14 urinary isolates from 2004) and animal (all faecal, 1 cattle, 1 chicken, 5 pigs, 9 cats, 1 dog, 1 rodent from 2008 to 2010) sources. In conclusion, this study highlights the importance of the IncFII group, pHK01-like plasmids in the dissemination of CTX-M-14 among isolates from diverse sources.     

spa typing and enterotoxin gene profile of Staphylococcus aureus isolated from bovine raw milk in Korea

Staphylococcus aureus is a major etiological pathogen of bovine mastitis, which triggers significant economic losses in dairy herds worldwide. In this study, S. aureus strains isolated from the milk of cows suffering from mastitis in Korea were investigated by spa typing and staphylococcal enterotoxin (SE) gene profiling. Forty-four S. aureus strains were isolated from 26 farms in five provinces. All isolates grouped into five clusters and two singletons based on 14 spa types. Cluster 1 and 2 isolates comprised 38.6% and 36.4% of total isolates, respectively, which were distributed in more than four provinces. SE and SE-like toxin genes were detected in 34 (77.3%) isolates and the most frequently detected SE gene profile was seg, sei, selm, seln, and selo genes (16 isolates, 36.3%), which was comparable to one of the genomic islands, Type I νSaβ. This is a first report of spa types and the prevalence of the recently described SE and SE-like toxin genes among S. aureus isolates from bovine raw milk in Korea. Two predominant spa groups were distributed widely and recently described SE and SE-like toxin genes were detected frequently.     

Epidemiology of extended spectrum beta-lactamase E. coli (CTX-M-15) on a commercial dairy farm

The epidemiology of an extended spectrum beta-lactamase Escherichia coli (CTX-M-15) was observed and described on a commercial dairy farm located in the United Kingdom. During 2008 longitudinal sampling of faecal pat samples from different cattle groups comprising milking and non-milking cows, calving cows, calves, and the environment was carried out. The proportion of CTX-M-15 E. coli positive samples was significantly (p<0.0.01) higher in milking cows (30.3%, CI(95%) 26.8; 33.8) than in the herd as a whole (17.0%, CI(95%) 14.9; 19.0). In 2008 95.6% of sampled calves tested positive for CTX-M-15 E. coli at two days of age. A more detailed investigation in 2009 revealed that cows and heifers were approximately eight times more likely to test positive in the 10 days after calving than the 9 days before (OR 7.6, CI(95%) 2.32; 24.9). The CTX-M15 E. coli was also readily isolated from the immediate calving pen environment, including the water troughs. A cyclic pattern was apparent where cows immediately after calving and as high yielders were highly positive, but where the prevalence decreased during the dry period. The increased prevalence of the CTX-M-15 E. coli in certain cattle groups and farm environments including calving pens suggested that husbandry, antimicrobial usage and hygiene may play a significant role on a farm with regards to the epidemiology of CTX-M-15. This may offer a practical opportunity to reduce further dissemination through good practice and hygiene around calving.     

Analysis of milk-derived isolates of E. coli indicating drug resistance in central Ethiopia

Mastitis is one of the most important diseases in dairy cows throughout the world and is responsible for significant economic losses to the dairy industry. This study was performed to characterize the genetic basis of drug resistance in Escherichia coli isolated from cases of clinical and sub-clinical bovine mastitis. A total of 224 California mastitis test (CMT)-positive milk samples were collected from December 2015 to April 2016 to characterize the phenotypic and genetic basis of antimicrobial resistance in E. coli isolated from raw milk from dairy farms found in Burayu, Sebeta, and Holeta areas of Ethiopia. The prevalence of E. coli was 7.1% (16) and both phenotypic and molecular techniques were used to identify E. coli antimicrobial susceptibility trait. The most commonly observed phenotypic resistance was against ampicillin (68.7%), sulphamethazole-trimethoprim (50%), and streptomycin (25%). Multidrug resistance phenotypes were found in 11 of 16 (68.7%) of E. coli isolates. Tetracycline (tet (A)) and chloramphenicol (cml (A)) genes were the most predominant encoding resistance genes identified (50%) each, followed by gentamycin resistance encoding gene (aac (3)-IV) (37.5%). Overall, 11 (68.7%) of the isolates had multidrug resistance genes responsible to two or more classes of antibiotics. The most common pattern detected was cml (A) and tet (A) together 37.5% followed by aac (3)-IV and tet (A) 25%. The current study indicated that raw milk could be regarded as critical source of antibiotic-resistant pathogenic E. coli.

     

Detection of Mycobacterium avium subsp. paratuberculosis in milk from clinically affected cows by PCR and culture

Milk and faeces samples from cows with clinical symptoms of paratuberculosis were examined for the presence of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) by culture and PCR. M. paratuberculosis was cultivated in variable numbers from faeces or intestinal mucosa in eight of 11 animals. In milk from five cows (all faeces culture positive), we cultivated a few colonies of M. paratuberculosis (<100 CFU per ml). Milk samples from two cows were PCR positive (both animals were faeces culture positive, and one cow was milk culture positive). One cow was culture negative on intestinal mucosa, but culture positive in milk, and two cows were negative in culture and PCR from both faeces and milk. In conclusion, the presence of M. paratuberculosis could be detected in raw milk by PCR, but cultivation of milk was more sensitive.     

Characterization of Staphylococcus aureus Isolated from Dairy Animals in Ireland

Enterotoxigenic Staphylococcus aureus frequently contaminate milk and milk products causing food poisoning. Staphylococcus aureus were isolated from bovine, ovine and caprine milk and milk filters from 78 dairy production holdings supplying the farmhouse cheese sector in Ireland, using standard culture methods. Molecular methods were applied to study the distribution of genes encoding staphylococcal enterotoxins and toxic shock syndrome toxin in the collection. Multilocus variable number tandem repeat analysis was used to subtype the collection. One hundred and two Staphylococcus aureus (54 milk filters and 48 bulk milk) were recovered from apparently healthy animals; half of the isolates were toxigenic. Our findings are discussed in light of the risks posed to public health.     

Abattoir-based study on the epidemiology of caprine tuberculosis in Ethiopia using conventional and molecular tools

Background

Despite the important role of goats for meat and milk production in Ethiopia, little information is available on the epidemiology of caprine tuberculosis (TB). Caprine TB is important as milk is usually consumed raw particularly by Ethiopian pastoralists. The objectives of the present study were to estimate the prevalence of TB in goats at an abattoir, to evaluate associated risk factors and to characterize the causative mycobacteria.

Methods

A cross-sectional study was conducted on 1990 randomly selected male goats that were slaughtered at Luna Export Abattoir of central Ethiopia. Postmortem examination, mycobacterial culturing and molecular typing techniques like genus typing, deletion typing and spoligotyping were used.

Result

The overall prevalence of caprine TB-like lesions was 3.5%. The lesion prevalence increased significantly with increasing age. Mycobacteria were found by culture and seen as acid fast bacilli in 12% of the goats with TB-like lesions. Characterization of the eight isolates using multiplex polymerase chain reaction (PCR) indicated that five of them belonged to the genus Mycobacterium. Four of the latter were confirmed to be members of the M. tuberculosis complex. Further characterization of the three M. tuberculosis isolates by spoligotyping identified them as type SIT53 and two new spoligotypes.

Conclusion

The isolation of M. tuberculosis from goats in this study indicates a potential risk of transmission of M. tuberculosis between humans and goats.     

Characteristics of ciprofloxacin and cephalosporin resistant Escherichia coli isolated from turkeys in Great Britain

1. A field study was performed to investigate the presence and characteristics of ciprofloxacin-resistant, extended spectrum β-lactamase (ESBL) and AmpC Escherichia coli from turkeys in Great Britain. E. coli were isolated from ～9000 boot swab samples from 27 different farms owned by four different companies. Between 1 and 14 visits were made to each farm (mean 3) at between 0 and 15?m intervals (mean?～?5?m).

2. CHROMagar ECC with and without ciprofloxacin or cephalosporin antibiotics was used as selective isolation media. Representative isolates with different phenotypes were tested for mutations in gyrA and for: qnrA, B, S, qepA and aac(6′)-Ib genes, for ESBL phenotype, the presence of bla _CTX-M genes and plasmid type, and for ampC genes_. Representative ciprofloxacin-resistant and CTX-M isolates were further tested for serotype and PFGE type. On ciprofloxacin selective media 55% of samples yielded ciprofloxacin resistant E. coli and of those further analysed, most had ciprofloxacin MICs >4 mg/l and mutations in gyrA.

3. For the different companies, the mean number of samples per farm with cefoxitin- or cefotaxime-resistant isolates ranged from 1·0% to 61·9% and 4·7% to 31·7% respectively. Cefotaxime-resistance was most commonly associated with an ESBL phenotype, a CTX-M-1 or CTX-M-14 sequence type and an I1-γ or K plasmid inc type. The mechanism of cefoxitin resistance was not determined for most isolates, but where determined it was bla _CMY-2.

4. PFGE and serotyping showed clonally-related isolates persisting over multiple visits suggesting both more prudent use of antibiotics and improved farm hygiene are needed to address the issue of antimicrobial resistance in isolates from turkeys.     

‘Nano-immuno test’ for the detection of live <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subspecies <Emphasis Type="Italic">paratuberculosis</Emphasis> bacilli in the milk samples using magnetic nano-particles and chromogen

Early rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP) bacilli in milk samples is the major challenge since traditional culture method is time consuming and laboratory dependent. We report a simple, sensitive and specific nano-technology based ‘Nano-immuno test’ capable of detecting viable MAP bacilli in the milk samples within 10 h. Viable MAP bacilli were captured by MAP specific antibody-conjugated magnetic nano-particles using resazurin dye as chromogen. Test was optimized using true culture positive (10-bovine and 12-goats) and true culture negative (16-bovine and 25-goats) raw milk samples. Domestic livestock species in India are endemically infected with MAP. After successful optimization, sensitivity and specificity of the ‘nano-immuno test’ in goats with respect to milk culture was 91.7% and 96.0%, respectively. Whereas, it was 90.0% (sensitivity) and 92.6% (specificity) with respect to IS900 PCR. In bovine milk samples, sensitivity and specificity of ‘nano-immuno test’ with respect to milk culture was 90.0% and 93.7%, respectively. However, with respect to IS900 PCR, the sensitivity and specificity was 88.9% and 94.1%, respectively. Test was validated with field raw milk samples (goats-258 and bovine-138) collected from domestic livestock species to detect live/viable MAP bacilli. Of 138 bovine raw milk samples screened by six diagnostic tests, 81 (58.7%) milk samples were positive for MAP infection in one or more than one diagnostic tests. Of 81 (58.7%) positive bovine raw milk samples, only 24 (17.4%) samples were detected positive for the presence of viable MAP bacilli. Of 258 goats raw milk samples screened by six diagnostic tests, 141 (54.6%) were positive for MAP infection in one or more than one test. Of 141 (54.6%) positive raw milk samples from goats, only 48 (34.0%) were detected positive for live MAP bacilli. Simplicity and efficiency of this novel ‘nano-immuno test’ makes it suitable for wide-scale screening of milk samples in the field. Standardization, validation and re-usability of functionalized nano-particles and the test was successfully achieved in field samples. Test was highly specific, simple to perform and easy to read by naked eyes and does not require laboratory support in the performance of test. Test has potential to be used as screening test to estimate bio-load of MAP in milk samples at National level.