Similar cefoxitin-resistance plasmids circulating in Escherichia coli from human and animal sources

The aim of this study was to determine the molecular epidemiology of cefoxitin-resistance Escherichia coli identified in cattle entering feedlots and determine if there were any similarities to E. coli causing human infections in Canadian hospitals. A total of 51 E. coli were isolated from a total of 2483 cattle entering four feedlots in southern Alberta, Canada. DNA fingerprinting using pulsed-field gel electrophoresis revealed thirty-two unique patterns with two major clusters observed comprised of Cluster A (11 strains) and Cluster B (7 strains). PCR and sequence analysis revealed 38 isolates (74.5%) harboured bla(CMY-2), whereas the remainder were found to contain mutations in the promoter region of the chromosomal ampC gene, which has been previously associated with cefoxitin resistance. No resistance to nalidixic acid, ciprofloxacin, or amikacin was observed in the clinical isolates. bla(CMY-2) harbouring plasmids were transferred to E. coli DH10B. All of the plasmids carrying bla(CMY-2) contained the A/C replicon and also harboured other resistance genes. Plasmid fingerprinting using BglII revealed 17 unique patterns with all but one clustering within 70% similarity. Comparison of the plasmid fingerprints to those isolated from human clinically significant E. coli in Canada during a similar time period [Mulvey, M.R., Bryce, E., Boyd, D.A., Ofner-Agostini, M., Land, A.M., Simor, A.E, Paton, S., 2005. The Canadian Hospital Epidemiology Committee, and The Canadian Nosocomial Infection Surveillance Program, Health Canada. Molecular characterization of cefoxitin resistant Escherichia coli from Canadian hospitals. Antimicrob. Agents Chemother. 49, 358-365] revealed four strains that harboured bla(CMY-2) A/C replicon type plasmid with fingerprint similarities of greater than 90% to the ones identified in E. coli from the cattle in this study. These findings highlight the potential linkage of multidrug resistant organisms in food producing animals and human infections in Canadian hospitals. The plasmids conferred resistance to multiple antibiotics which could limit options for the treatment of infections caused by these strains.     

Molecular and phenotypic characteristics of CMY-2 β-lactamase-producing Salmonella enterica serovar Typhimurium isolated from cattle in Japan

Isolates of extended-spectrum cephalosporin (ESC)-resistant Salmonella enterica serovar Typhimurium obtained from two different farms in Fukushima Prefecture, Japan, in 2007 were characterized in order to determine the genetic basis of resistance. ESC resistance in the two isolates was mediated by an AmpC β-lactamase encoded by the bla(CMY-2) gene, which is located in a large self-transmissible plasmid in each isolate. The sizes of the bla(CMY-2)-carrying plasmids were different. The replicon types of the plasmids were I1-Iγ and A/C. The results of macrorestriction analysis and phage typing suggest a close relationship between both isolates. This is the first report of ESC-resistant S. Typhimurium isolated from cattle in Japan.     

Faecal shedding of CTX-M-producing Escherichia coli in horses receiving broad-spectrum antimicrobial prophylaxis after hospital admission

The objective of this longitudinal study was to investigate the occurrence and genetic background of faecal Escherichia coli resistant to cefotaxime (CTX) in horses receiving broad-spectrum antimicrobial prophylaxis after admission to a veterinary teaching hospital. The ten horses enrolled in the study were treated with cefquinome either alone (n=4) or in combination with metronidazole (n=3) or other antimicrobial agents (n=3). CTX-resistant coliforms in faeces collected before, during and after treatment were quantified on selective MacConkey agar supplemented with CTX, and a colony isolated randomly from each positive sample was characterized by pulsed-field gel electrophoresis, and by PCR detection and sequencing of bla(TEM), bla(SHV), bla(CTX-M) and bla(CMY). All horses were negative for CTX-resistant coliforms at admission but became positive within the first three days of treatment. The average faecal densities of CTX-resistant coliforms increased significantly following antimicrobial prophylaxis (P<0.001). Genetic characterization of 29 faecal isolates revealed that this effect was due to proliferation of E. coli producing either CTX-M-1 (n=28) or CTX-M-14 (n=1). Five CTX-M-1 isolates produced additional β-lactamases (TEM-1, CMY-34 and the novel variant CMY-53). Shedding of CTX-M-producing E. coli appeared intermittent in four horses and persisted two weeks after antimicrobial treatments in five of six patients tested after discharge from hospital. Nosocomial transmission was suggested by finding five identical CTX-M-1-producing E. coli pulsotypes in multiple horses. The originality of the study lies in the unanticipated high frequency and genetic diversity of CTX-M-producing E. coli observed in the faecal flora of hospitalized patients receiving broad-spectrum antimicrobial prophylaxis.     

Prevalence of antimicrobial-resistant Escherichia coli in dogs in a cross-sectional, community-based study

The prevalence of carriage of antimicrobial-resistant (AMR) and extended-spectrum β-lactamase (ESBL)-producing Escherichia coli was determined in 183 healthy dogs from a semi-rural community in Cheshire. Isolates were tested against a panel of antimicrobials and by PCR to detect resistance genes. In the suspected ESBL-producing isolates, the presence of bla(SHV), bla(TEM), bla(CTX-M) and bla(AmpC) genes was determined by PCR and sequencing. A total of 53 (29 per cent, 95 per cent confidence interval [CI] 22.4 to 35.5 per cent) dogs carried at least one AMR E coli isolate. Twenty-four per cent (95 per cent CI 17.9 to 30.2 per cent) of dogs carried isolates resistant to ampicillin, 19.7 per cent (95 per cent CI 13.9 to 25.4 per cent) to tetracycline and 16.9 per cent (95 per cent CI 11.5 to 22.4 per cent) to trimethoprim. A bla(TEM) gene was detected in 39 of 54 ampicillin-resistant isolates, a tet(B) gene in 12 of 45 tetracycline-resistant isolates, and a dfr gene in 22 of 33 trimethoprim-resistant isolates. Multidrug-resistant isolates were demonstrated in 15 per cent (28 of 183; 95 per cent CI 10.1 to 20.5 per cent) of dogs. Nine suspected ESBL-producing E coli were isolated, of which only one was confirmed by double disc diffusion testing. Two of these isolates carried the bla(TEM-1) gene and seven carried the bla(CMY-2) gene.     

Characteristics of extended-spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae isolates from horses

The aim of the present study was to contribute to the knowledge on extended-spectrum beta-lactamases (ESBL's), AmpC beta-lactamases and integrons in Enterobacteriaceae isolated from horses, which is still limited. The susceptibility of 1581 clinical isolates from animals to ceftiofur was tested. Most of these isolates (n=1347) originated from horses. Seven ceftiofur-resistant equine isolates (four Escherichia coli and three Klebsiella pneumoniae) were identified and all seven were multidrug-resistant. These isolates were further studied for the presence of ESBL's, AmpC beta-lactamases and class 1 integrons. The potential for the horizontal transfer of resistance genes among these clinical isolates was also studied. ESBL-type resistance genes were found in five isolates, AmpC-type genes in one isolates and integrons in six isolates. Nucleotide sequence analysis revealed that the isolates carried the bla(CTX-M-1), bla(CMY-2), bla(TEM-1) and/or bla(SHV-1) genes. This is the first report describing the in vitro conjugal transfer of the bla(CTX-M-1) genes from a clinical E. coli isolate to Salmonella isolates. Gene cassettes encoding resistance to aminoglycosides (aadA1, aadA2 and aadA5), and trimethoprim (dfrA1, drfA12 and dfrA17) were found on the integrons present in the isolates. The cassette arrays of the dfrA17-aadA5 and dfrA1-aadA1 genes in the two integrons of a single E. coli isolate have not yet been described before. To our knowledge this is the first report on ESBL's and AmpC beta-lactamases in equine E. coli and Klebsiella isolates.     

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。     

Efficacy and tolerability of once-daily cephalexin in canine superficial pyoderma: an open controlled study

OBJECTIVES: The aims of this study were to evaluate the efficacy and tolerability of oral cephalexin given at 30 mg/kg once daily in dogs with superficial pyoderma and to compare them with those of oral cephalexin given at 15 mg/kg twice daily. METHODS: Twenty dogs with superficial pyoderma were treated with cephalexin at 30 to 60 mg/kg orally once daily (group A) and compared with 20 dogs treated at a dose of 15 to 30 mg/kg orally twice daily (group B). Dogs were treated until 14 days after clinical remission. Type and distribution of lesions, pruritus and general health status were assessed every 14 days using a numerical scale until 14 days after treatment discontinuation. Total scores for each evaluation day were compared between the two groups as well as time to obtain resolution and percentage of relapses. RESULTS: Resolution of superficial pyoderma was obtained in all dogs in 14 to 42 days (median 28 days for both groups), with no difference between groups. Six dogs experienced vomiting or diarrhoea but did not require discontinuation of the treatment. Only one dog (in group A) relapsed nine days after treatment discontinuation. CLINICAL SIGNIFICANCE: Once-daily cephalexin is as effective as twice-daily cephalexin in the treatment of canine superficial pyoderma.     

Effects of an ethyl lactate shampoo in conjunction with a systemic antibiotic in the treatment of canine superficial bacterial pyoderma in an open-label,nonplacebo-controlled study

An open-label, nonplacebo-controlled study was designed to compare systemic cephalexin therapy versus systemic cephalexin and ethyl lactate shampoo therapy in the treatment of canine superficial bacterial pyoderma. Twenty client-owned dogs diagnosed with generalized superficial bacterial pyoderma (SP) were alternately assigned to oral treatment with cephalexin (25 to 30 mg/kg every 12 hours) or treatment with cephalexin (as for Group 1) and twice-weekly shampooing with a 10% ethyl lactate shampoo, which was left in contact with the dog's skin for 10 minutes. On Days 14 and 28, skin lesion severity scores, assessed by the investigators, were significantly (P <.01) lower for the group treated with cephalexin and shampoo than for the group treated with cephalexin only. On Day 14, dog owners gave better scores to dogs treated with cephalexin and shampoo for haircoat appearance and body odor than for dogs treated only with cephalexin. Clinical and cytologic resolution of SP occurred significantly (P <.02) sooner in the cephalexin/shampoo group (29.4 days) than in the cephalexin only group (37.8 days).     

Prevalence of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in food-producing animals

To evaluate the diversity of extended-spectrum β-lactamases (ESBL) genes among food-producing animals, 48 isolates of ESBL-producing Escherichia coli isolates were obtained from rectal samples of broilers, layers, beef cattle and pigs, at the slaughterhouse level. ESBL-carrying E. coli were isolated from 60.0% of individual broiler rectal samples, 5.9% of layers, 12.5% of beef cattle and 3% of pigs. One ESBL-producing Klebsiella pneumoniae was isolated from a broiler. The ESBL-positive E. coli isolates from broilers harbored various ESBL genes: bla (SHV-12), bla(CTX-M-2), bla(CTX-M-14), bla(CTX-M-15) and bla(CTX-M-44). The plasmid DNAs were analyzed by restriction patterns. Homogeneous band patterns were yielded in those of K. pneumoniae and E. coli isolates harboring the bla(CTX-M-2) gene from different farms. No genetic relation between the 2 CTX-M-14 ESBL-producing strains was found by pulsed-field gel electrophoresis, although 2 plasmids in these strains, obtained from different broiler farms, were similar to each other. This study provides evidence that the proliferation of CTX-M-producing E. coli is due to the growth of indigenous CTX-M-producing strains and the possible emergence of strains that acquired CTX-M genes by horizontal transfer in different broiler farms. CTX-M-producing coliforms in broilers should be controlled due to the critical importance of cephalosporins and the zoonotic potential of ESBL-producing bacteria.     

Antimicrobial resistance in Staphylococcus spp., Escherichia coli and Enterococcus spp. in dogs given antibiotics for chronic dermatological disorders, compared with non-treated control dogs

The aim of this study was to evaluate antimicrobial resistance in canine staphylococci, Escherichia coli and enterococci, which were isolated from 22 dogs with pyoderma and a history of previous antibiotic treatment, compared to bacterial isolates from 56 non-treated control dogs. Two isolates of each bacterial species per dog were investigated, if detected. Staphylococcal isolates from dogs with pyoderma (35 isolates) were more resistant to sulphatrimethoprim than the isolates from controls (56 isolates) (57% vs. 25%, p < 0.004). Multiresistance in staphylococci was also more common in dogs with pyoderma (29% vs. 9%, p = 0.02). A similar trend among isolates of E. coli was detected (24 and 74 isolates from treated and control dogs, respectively), but the differences were not significant. Resistance for macrolide-lincosamides was approximately 20% among staphylococci in both groups. Resistance to ampicillin among enterococci was 4%-7%. The age of the dogs might have an impact on resistance: multiresistance among staphylococcal isolates from younger dogs (< or = 5 years) was more common than in older dogs (26 years) (24%, vs. 0%, 63 and 27 isolates, respectively, p = 0.02). Staphylococci in younger dogs were more resistant to tetracycline (48% vs. 11%, p < 0.001) and sulphatrimethoprim (48% vs. 15%, p < 0.01) than those in older dogs. In contrast, the isolates of E. coli from older dogs tended to be more resistant, although a significant difference was detected only in resistance to tetracycline (13% vs. 2% of 40 and 50 isolates respecthely, p = 0.04)). The results of this small study indicate that resistance in canine staphylococci in the capital area of Finland is comparable with many other countries in Europe. Resistance in indicator bacteria, E. coli and enterococci, was low.     

动物源产CMY-2大肠杆菌分子流行病学研究

为调查产CMY-2大肠杆菌在广东各养殖场的流行情况,对2010—2011年间分离自猪、鸡、鸭、鹅等动物的1293株大肠杆菌,采用PCR方法筛选出bla_CMY-2阳性菌株,琼脂稀释法测定阳性菌株对17种抗微生物药物的敏感性;接合转移试验和XbaⅠ酶切PFGE图谱分析bla_CMY-2基因转移扩散的方式。结果显示,1293株大肠杆菌中27 株含有bla_CMY-2 基因,检出率为2.09%,均为多重耐药菌株,主要耐药谱型为AMP/CHL/TET/FLF/CTF/CTX/CAZ/CTR/GEN/CIP/ENR/NAL/OQX;27 株携带bla_CMY-2 基因菌株中有14 株的bla_CMY-2 基因可随质粒转移到受体菌E.coli C600中,且往往与bla_TEM-1和(或)qnrS1共同转移;PFGE分析结果显示,27 株携带bla_CMY-2 基因菌株共产生17条谱带,其中有4株菌株,两两分别来自同一地区,存在克隆传播关系。提示,在广东地区食品动物养殖场内存在产CMY-2大肠杆菌的克隆传播,且bla_CMY-2 基因伴随可转移质粒或其他可转移移动元件可能是造成产CMY-2大肠杆菌流行分布的主要原因。     

Prospective study of bacterial overgrowth syndrome in eight dogs

Eight dogs with cutaneous lesions, clinical signs and cytological findings compatible with bacterial overgrowth syndrome were compared with four healthy dogs. The affected dogs were treated for 28 days with 30 mg/kg/day cephalexin. The results showed that the syndrome was a superficial cutaneous disorder characterised by marked pruritus, greasy seborrhoea, offensive odour, erythema, lichenification, hyperpigmentation, excoriations and alopecia involving principally the ventral aspect of the body, but no papules, pustules, epidermal collarettes or crusts; it was caused by overgrowths of Staphylococcus intermedius all over the body surface. Histopathological findings included a superficial, perivascular, hyperplastic and spongiotic dermatitis with a mixed inflammatory infiltrate, but no lesions suggestive of a true pyoderma. In the affected dogs, anti-staphylococcal immunoglobulin G levels were high, but anti-staphylococcal immunoglobulin E levels were low, suggesting that staphylococcal hypersensitivity is not the underlying pathogenic process. The antibiotic treatment improved the condition of all the dogs, but five of the eight had an underlying allergic skin disease.     

广东地区患病食品动物源大肠杆菌ESBLs和AmpC酶流行分布调查

质粒介导的超广谱β-内酰胺酶（ESBLs）和AmpC酶广泛传播和扩散,导致全球范围内肠杆菌科细菌对超广谱头孢菌素类的耐药性发展迅速,引起人们高度重视。食品动物源大肠杆菌可作为耐药基因储库,对细菌耐药性在动物、环境和人之间的传播起着非常重要的作用。本研究从2009年患病的食品动物,包括鸡、鸭、鹅和猪,分离鉴定了315株大肠杆菌,调查ESBLs和AmpC酶在患病食品动物中的流行分布。经双纸片协同扩散实验筛选出61株ESBL阳性菌株,PCR检测共检测出8种blaCTX-M基因,分别为blaCTX-M-14/14b、blaCTX-M-79、blaCTX-M-65、blaCTX-M-27、blaCTX-M-15、blaCTX-M-24、blaCTX-M-98和blaCTX-M-13,其中检出率最高的为blaCTX-M-79,其次为blaCTX-M-14/14b。本研究还检测出1株新的SHV型酶,命名为SHV-135;PCR共检测出5株大肠杆菌携带质粒源CMY-型AmpC酶,其中2株鹅源大肠杆菌携带1个新型CMY酶,命名为CMY-64。本研究表明患病动物分离的大肠杆菌中ESBLs和AmpC酶存在复杂性和多样性,而且新型β-内酰胺酶检出日益增多,提示兽医临床应慎用β-内酰胺类抗生素。     

Pyoderma caused by Pseudomonas aeruginosa infection in dogs: 20 cases

In this report we describe the historical, clinical, histopathological and microbiological features, as well as treatments and clinical outcome, of pyoderma where Pseudomonas aeruginosa alone was isolated on bacterial culture from lesional skin. Twenty dogs were included in this retrospective study. Seven dogs without prior history of systemic or skin disease presented with acute deep pseudomonal pyoderma characterized by a sudden onset of dorsal truncal pain. Skin lesions in these dogs consisted of erythematous papules, haemorrhagic bullae, ulcers and haemorrhagic crusts confined to the dorsum. An excellent clinical response was achieved with 3-4 weeks of treatment with oral fluoroquinolones. Thirteen dogs with a more gradual onset of skin lesions associated with pseudomonal pyoderma had a history of prior skin, ear or systemic disease and had previously been treated with antibiotics and/or immunomodulatory drugs. Skin lesions in these dogs were variable and similar to those described for superficial and deep staphylococcal pyoderma. In this group, one dog was euthanized prior to commencement of treatment, two dogs were lost to follow up, and 9 had resolution of lesions following treatment with topical silver sulfadiazine (one dog), fluoroquinolones (six dogs) or cephalexin (two dogs) administered orally for 3 to 12 weeks. Rod-shaped bacteria were not always detected on cytology. Histopathology of dogs with deep pseudomonal pyoderma was characterized by severe perforating suppurative folliculitis and furunculosis.     

Occurrence of antimicrobial resistant bacteria in healthy dogs and cats presented to private veterinary hospitals in southern Ontario: A preliminary study

The prevalence and patterns of antimicrobial susceptibility of fecal Escherichia coli, Salmonella spp., extended β-lactamase producing E. coli (ESBL-E. coli), methicillin-resistant Staphylococcus aureus (MRSA), and methicillin-resistant Staphylococcus pseudintermedius (MRSP) were determined for healthy dogs (n = 188) and cats (n = 39) from veterinary hospitals in southern Ontario that had not had recent exposure to antimicrobials. The prevalence of antimicrobial resistance in E. coli was as follows: streptomycin (dogs — 17%, cats — 2%), ampicillin (dogs — 13%, cats — 4%), cephalothin (dogs — 13%, cats — < 1%), and tetracycline (dogs — 11%, cats — 2%). Eleven percent of dogs and 15% of cats had isolates that were resistant to at least 2 antimicrobials. Cephamycinase (CMY)-2 producing E. coli was cultured from 2 dogs. No Salmonella spp., ESBL-E. coli, MRSA, or MRSP isolates were recovered. The observed prevalence of resistance in commensal E. coli from this population was lower than that previously reported in companion animals, but a small percentage of dogs may be a reservoir for CMY-2 E. coli.     

Multiple antimicrobial resistance region of a putative virulence plasmid from an Escherichia coli isolate incriminated in avian colibacillosis

Infections due to Escherichia coli have been costly to the poultry industry, but the exact virulence mechanisms used by these organisms to cause disease in birds remain undefined. Several factors have been shown to contribute to the virulence of avian E. coli, and many of the genes encoding these factors have been found on large conjugative plasmids. Because of the occurrence of antimicrobial resistance genes on these same plasmids, it is possible that the use of antimicrobial agents may select for persistence of E. coli containing such plasmids. In the present study, a subclone of one of these plasmids was identified as likely containing some virulence and antimicrobial resistance genes. In an effort to better understand the relationship between virulence and resistance in these plasmids, this subclone was sequenced and the sequence analyzed. Analysis of this 30-kilobase (kb) region of plasmid pTJ100 revealed a mosaic of virulence genes, insertion sequences, antimicrobial resistance cassettes, and their remnants. Many of the resistance genes found in this region were expressed under laboratory conditions, indicating that certain antimicrobial agents, including disinfectants, antibiotics, and heavy metals, could promote selection of E. coli containing such plasmids in the production environment. Also, analysis of the G + C content of this clone indicated that it is the likely consequence of a complex evolution with components derived from various sources. The occurrence of many mobile elements in conjunction with antimicrobial resistance and virulence genes in this 30-kb region may indicate that the genetic constitution of the clone is quite plastic. Although further study will be required to better define this plasmid's role in avian E. coli virulence, the sequence described here is, to our knowledge, the longest known contiguous sequence of a ColV plasmid yet presented. Analysis of this sequence indicates that this clone and its parent plasmid may be important to the pathogenesis of avian colibacillosis and the evolution of avian E. coli virulence.     

Post-antibiotic effect of cephalexin against isolates of Staphylococcus intermedius obtained from cases of canine pyoderma

The post-antibiotic effect in vitro (PAE) of cephalexin was determined according to a broth dilution method against 5 isolates of Staphylococcus intermedius obtained from cases of canine pyoderma. Two durations of exposure and 3 concentrations were tested. The PAE increased when time of exposure or concentration increased. The mean PAE ranged from 0.7 to 3.3 h. The PAE of cephalexin against Staph-ylococcus intermedius may be clinically relevant when selecting a dosage regimen to treat pyoderma in dogs.     

Change in antimicrobial resistance pattern in Salmonella enterica serovar Typhimurium isolates detected in a beef cattle farm

Multidrug-resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates with four different antimicrobial resistance patterns obtained from a beef cattle farm were characterized to determine their clonality. Macrorestriction analysis of genomic DNA revealed that these four isolates are closely related to each other and can be classified as a newly emerged pulsed-field gel electrophoresis type among cattle: cluster VII. Three of the four isolates showed resistance to extended-spectrum cephalosporins (ESCs), and this resistance was mediated by AmpC β-lactamase encoded by the bla(CMY-2) gene in a 190-kbp IncA/C plasmid. Results of restriction analysis and IncA/C backbone PCR suggest that the three 190-kbp plasmids are identical and that a 70-kbp IncA/C plasmid of the ESC-susceptible isolate is derived from the 190-kbp plasmid by a deletion event. Three isolates harboured a virulence-resistance plasmid (165 or 180 kbp), and restriction analysis revealed that these plasmids were identical or closely related to each other. These results suggest that the four S. Typhimurium cluster VII isolates originate from a common ancestor that probably invaded the farm prior to the salmonellosis outbreak. Antimicrobial resistance patterns may not necessarily reflect the relationships of the isolates.     

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.     

Enterotoxin plasmids in bovine and porcine enterotoxigenic Escherichia coli of O groups 9, 20, 64 and 101.

Fourteen strains of Escherichia coli of serogroups characteristic of porcine class 2 enterotoxigenic E. coli isolated from pigs or calves were selected for genetic studies. The strains were examined for their ability to cotransfer a number of plasmid-mediated properties during conjugation with E. coli K-12. These properties were antibiotic resistance, and the production of heat-stable enterotoxin, the K99 antigen and colicin and the ability to ferment raffinose. Distinction was made between the two types of heat-stable enterotoxin, STa and STb. All 14 strains were antibiotic resistant and 11 of them cotransferred antibiotic resistance and heat-stable enterotoxin. One strain which transferred heat-stable enterotoxin also transferred the raffinose gene. Among six K99-positive strains which transferred heat-stable enterotoxin, five always cotransferred K99. Three strains had 100% cotransfer of colicin as well as heat-stable enterotoxin and K99. Drug resistance determinants were cotransferred at high frequency with heat-stable enterotoxin for six of eight multiple drug resistant enterotoxigenic E. coli. A 100% cotransfer of combinations of heat-stable enterotoxin, K99, colicin and antibiotic resistance was often associated with a single plasmid band on agarose gel electrophoresis. For some strains, the genes for STa and STb were on the same plasmid and for others they were on separate plasmids. The enterotoxin plasmids ranged in size from 5.2 to 85 Mdal. Heterogeneity in molecular size occurred among enterotoxin plasmids in E. coli of the same serogroup and recovered from the same animal host species.