Relationship between level of antibiotic use and resistance among Escherichia coli isolates from integrated multi-site cohorts of humans and swine

The objective of this longitudinal ecological study was to examine the relationship between the prevalence of antibiotic-resistant (AR) commensal Escherichia coli isolates from both monthly human wastewater and composite swine fecal samples and the concurrent aggregated monthly antibiotic use recorded within each host species in multi-site vertically integrated swine and human populations. In addition, human vocation (swine worker versus non-swine worker), swine production group, and season were examined as potential confounding variables. Human and swine E. coli isolates (n = 2469 human and 2310 swine, respectively) were tested for antimicrobial susceptibility using a commercial broth microdilution system. In the human population, among swine workers the relative odds of tetracycline resistance were increased significantly for tetracycline (class) drug use at the third quartile and above of mean monthly dosage (MMD) (OR = 1.8) as compared to the referent category (non-use). The relative odds of ciprofloxacin resistance were significantly increased for ciprofloxacin use in non-swine workers (OR = 5.5) as compared to the referent (non-use). The relative odds of tetracycline resistance were increased significantly for chlortetracycline use in medicated feed for the upper tertile of MMD category (OR = 2.9) as compared to the referent category (no use) across all swine production groups. While high variability among seasonal samples over the 3-year period was observed, no common seasonal trends relating to antibiotic use and prevalence of resistance over the 3-year period were apparent. The overall effects of concurrent human and swine antibiotic use on AR E. coli levels were inconsistent and modest in this study.     

牛肉源大肠杆菌的耐药性检测及相关耐药基因分布

为了解牛肉源大肠杆菌的耐药性并检测其相关耐药基因分布,本研究选取了117株牛肉源大肠杆菌,经药敏纸片法对11种抗菌药物进行了药敏检测,并根据耐药表型利用普通和(或)多重PCR技术对耐四环素菌中tet(A)、tet(B)和tet(C)基因,耐氨苄西林菌中blaTEM1、blaPSE1和blaOXA1基因,耐链霉素菌中strA-strB、aadA1基因,耐磺胺类药菌中sul1、sul2和sul3基因进行了调查分析。结果显示,117株大肠杆菌对四环素、氨苄西林、链霉素和磺胺异恶唑的耐药率较高,分别为89%、42%、38%和22%。tet(A)基因是所有四环素耐药基因中最为流行的一种基因(55%);在检测的3个β-内酰胺类药物耐药基因中,最流行的为blaTEM1基因(73%);链霉素的耐药性主要由strA-strB基因(38%)编码;sul2基因在耐磺胺异恶唑菌中的检出率最高(77%)。结果表明本次分离的牛肉源大肠杆菌耐药非常严重,进一步肯定了肉牛业生产中抗菌药的使用对大肠杆菌耐药性的产生和发展所发挥的重要作用,提示食品动物养殖应严格控制饲料中抗菌药的滥用。     

Detection of fluoroquinolone resistance level in clinical canine and feline Escherichia coli pathogens using rapid real-time PCR assay

Fluoroquinolones are used to treat infections caused by Escherichia coli in canine and feline veterinary patients, particularly those infecting the urinary tract. The gyrA gene is a primary target causing fluoroquinolone resistance in Gram negative coliforms, with mutations in codons 83 and 87 generally associated with high-level of resistance E. coli clinical isolates. We have developed a fluorescence resonance energy transfer (FRET) quantitative PCR to identify enrofloxacin-resistance in clinical E. coli isolates that carry mutations in codons 83 and 87 of gyrA. This real-time quantitative PCR assay is rapid, economical, and sensitive compared with cultured antimicrobial susceptibility testing. The assay identified as few as four genome copies per reaction from culture and 19 genome copies in urine. For the 70 isolates tested, the sensitivity was 87.5% (95% CI = 75–95.3%) (n = 42/48), specificity was 100% (95% CI = 87.3–100%) (n = 22/22), whereas accuracy was 91.4% (95% CI = 82.3–97%) (n = 64/70). Furthermore, we were able to accurately differentiate between the wild type and mutants E. coli directly from infected canine urine samples (n = 5) within 2 h. These results were confirmed by sequence alignments of the PCR products and comparison with the susceptibility testing. The FRET-PCR assay appears to have promising clinical application as an early diagnostic tool for rapid and sensitive detection and differentiation of the level of fluoroquinolone resistance among clinical E. coli isolates that may facilitate design of the dosing regimen.     

Molecular screening and risk factors of enterotoxigenic Escherichia coli and Salmonella spp. in diarrheic neonatal calves in Egypt

The aim of the present study was to carry out molecular epidemiological investigation on enterotoxigenic Escherichia coli (ETEC) K99 and Salmonella spp. in diarrheic neonatal calves. Fecal samples were obtained from 220 diarrheic calves at 9 farms related to four governorates in central and northern Egypt. E. coli and Salmonella spp. isolates were examined for E. coli K99 and Salmonella spp. using PCR. ETEC K99 was recovered from 20 (10.36 %) out of 193 isolates, whereas Salmonella spp. was recovered from nine calves (4.09%).Multivariable logistic regression was used to evaluate the risk factors associated with both infections. ETEC K99 was significantly affected by age (P < 0.01; OR: 1.812; CI 95%: 0.566–1.769), colostrum feeding practice (P < 0.01; OR: 5.525; CI 95%: 2.025–15.076), rotavirus infection (P < 0.001; OR: 2.220; CI 95%: 0.273–1.251), vaccination of pregnant dams with combined vaccine against rotavirus, coronavirus and E. coli (K99) (P < 0.001; OR: 4.753; CI 95%: 2.124–10.641), and vitamin E and selenium administration to the pregnant dam (P < 0.01; OR: 3.933; CI 95%: 0.703–1.248).Infection with Salmonella spp. was found to be significantly affected by the animal age (P < 0.05; OR: 0.376; CI 95%: 0.511–1.369), Hygiene (P < 0.05; OR: 0.628; CI 95%: 1.729–5.612), and region (P < 0. 01; OR: 0.970; CI 95%: 0.841–1.624).The results of the present study indicate the importance of PCR as rapid, effective and reliable tool for screening of ETEC and Salmonella spp. when confronted with cases of undifferentiated calf diarrhea. Moreover, identification of the risk factors associated with the spreading of bacteria causing diarrhea may be helpful for construction of suitable methods for prevention and control.     

Prevalences of resistance to seven antimicrobials among fecal Escherichia coli of swine on thirty-four farrow-to-finish farms in Ontario, Canada

Fecal specimens were composited and a hydrophobic-grid membrane-filter method was used to measure antimicrobial resistance to ampicillin 16 μg/ml, carbadox 30 μg/ml, gentamicin 4 μ/ml, nitrofurantoin 32 μg/ml, spectinomycin 16 μg/ml, sulfisoxazole 32 μg/ml and tetracycline 8 μg/ml among 8119 Escherichia coli isolates from 68 fecal samples collected on 34 farrow-to-finish swine farms marketing over 500 hogs/yr. The overall prevalences of resistance to antimicrobials among these isolates were: ampicillin 29%, carbadox 3.5%, gentamicin 0.6%, nitrofurantoin 27%, spectinomycin 28%, sulfasoxizole 38% and tetracycline 71%. Thirty to seventy-six per cent of the variations in prevalences were explained by between-farm differences.     

Field trial evaluating changes in prevalence and patterns of antimicrobial resistance among Escherichia coli and Enterococcus spp. isolated from growing broilers medicated with enrofloxacin, apramycin and amoxicillin

The present study investigates, under field conditions, the influence of antimicrobial administration on prevalence and patterns of antimicrobial resistance among Escherichia coli and Enterococcus spp. isolated from growing broilers. For this purpose, a group of 16,000 commercial broiler chickens was treated with enrofloxacin from day 1 to day 3, gentamicin from day 19 to day 21, and ampicillin from day 26 to day 28. A control group of 16,000 broilers was placed in the same controlled environment poultry house. Fecal (from both groups) and feed samples were collected at regular intervals. Few E. coli isolates were obtained from either farm environment or poultry feed samples, while enterococci were found to be ubiquitous among these samples. The frequency of resistance against most antimicrobials tested was significantly higher (P < 0.05) in E. coli isolated from broilers receiving intermittent antimicrobial pressure than that from non-medicated broilers, whereas in enterococci these differences were only observed among structurally related antimicrobial drugs and over a short period of time. By the time the broilers reached market age (33 days), several multi-resistant E. coli and enterococci were detected in the feces of the medicated group. Results suggest that antimicrobial resistance in E. coli was mainly medication-dependent, whereas among enterococci, changes observed over time were apparently influenced by factors apart from antimicrobial exposure, namely the resistance organisms previously present in farm environment and those present in feedstuffs.     

Prevalence of Multi-Drug-Resistant Salmonella in Equids Maintained by Low Income Individuals and on Designated Equine Farms in India

We examined 872 equids (445 maintained by low-income individuals and 427 maintained on nine designated equine farms) and, using previously described methods for bacteria, isolated Salmonella from fecal samples of 59 (6.77%) animals. Of the 646 horses, 183 donkeys, and 43 mules that had feces cultured for Salmonella, 42 (6.5%), 7 (3.8%), and 10 (23.3%), respectively, were excreting Salmonella strains in feces. Six horse mares were excreting Salmonella enterica of two different serovars simultaneously. A total of 65 Salmonella enterica isolates belonged to 13 serovars, namely S. paratyphi B var Java (14), S. I. 4, 5, 12, 27: r, i: 1, 5 (11), S. Drogana (8), S. Newport (7), S. Saintpaul (5), S. Lagos (4), S. Typhimurium (5), S. Kottbus (3), S. Bovismorbificans (3), S. Dumfries (2), S. Tshiongwe (1) S. Weltevreden (monophasic) (1), and S. enterica ssp salamae (1). With Salmonella-specific polymerase chain reaction (PCR) using hisJ gene primers, 107 (12.3) fecal samples yielded a specific amplicon of 496 bp. On using PCR, prevalence of Salmonella in donkeys, horses, and mules was 4.9%, 10.8%, and 65.1%, respectively. With both methods of Salmonella detection in feces, prevalence was significantly higher in female than in male donkeys and horses. Salmonella shedding in feces was significantly higher in equids maintained by low-income people than those at designated equine farms. Almost all Salmonella isolates (63 of 65) had multiple-drug-resistance (MDR, resistance to three or more drugs). Salmonella isolates were commonly resistant to sulfamethoxazole (90.8%), tetracycline (70.8%), doxycycline (67.7%), furazolidone (66.2%), and colistin (55.4%). A few isolates had resistance to trimethoprim (3.1%), ciprofloxacin (3.1%), ceftriaxone (3.1%), ceftazidime (3.1%), cefoperazone (3.1%), chloramphenicol (4.6%), cefotaxime (6.2%), gentamicin (9.2%), ampicillin + cloxacillin (9.2%), cotrimoxazole (13.8%), kanamycin (13.8%), amoxicillin + clavulanic acid (16.9%), imipenem (16.9%), ampicillin (18.5%), amikacin (23.1%), neomycin (27.7%), nalidixic acid (33.8%), and streptomycin (36.9%). With the exception of 13 Salmonella isolates of S. Drogana (4), S. Newport (4), S. I. 4, 5, 12, 27: r, i: 1, 5 (4) and S. Kottbus (1) serovars, all had one or more than one plasmid. Molecular weight of plasmids ranged between 3 kDa and >87 kDa. One heavy plasmid (≥87 kda) was present in all the 52 plasmid-positive strains. Presence of plasmid could not be correlated with MDR in Salmonella isolates from equids.     

Molecular and virulence characteristics of multi-drug resistant Salmonella Enteritidis strains isolated from poultry

Forty-six Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) strains were isolated from chicken meat, faeces, and eggshells collected from hatcheries throughout Korea. The strains were examined for the presence of antimicrobial resistance and virulence genes. All 46 isolates were resistant to at least one of 21 antibiotics used in this study, 30 (65.2%) were resistant to three or more antimicrobials, and a single remarkable isolate was resistant to 15 antimicrobials. The isolates were primarily resistant to penicillins, sulfisoxazole, streptomycin, tetracycline and quinolones.The high rate of resistance in S. Enteritidis strains, sometimes to multiple drugs, may complicate future options for treating human infections. Nineteen of the 21 penicillin resistant isolates carried the bla_TEM gene, while one strain, resistant both to penicillins and ceftriaxone, carried the bla_CTX-M gene. Thirty-seven of the 45 sulfisoxazole resistant isolates carried sul2, and 23/24 streptomycin resistant isolates carried both strA and strB. All 10 tetracycline resistant isolates carried the tet(A) gene. Most isolates harboured both SPI-1 and SPI-2-associated genes, and the spv operon, which are known to be associated with human infections. The presence of these genes suggests that these strains could give rise to public health problems if dispersed in the general human population.     

Identification of the tet(B) resistance gene in Streptococcus suis

The tetracycline resistance gene, tet(B), has been described previously in Gram negative bacteria. In this study tet(B) was detected in plasmid extracts from 17/111 (15%) Streptococcus suis isolates from diseased pigs, representing the first report of this resistance gene in Gram positive bacteria.     

四川省部分地区山羊沙门氏菌健康带菌率及耐药性调查

为了解四川省山羊沙门氏菌带菌情况,本试验采集了四川省部分地区5个规模化山羊养殖场表观健康山羊粪便196份,经BPW预增菌、TTB选择性增菌后,采用靶向invA基因的PCR方法检测沙门氏菌的带菌率;对阳性样本进行细菌分离鉴定,随机选择25株沙门氏菌分离株测定其对15种抗菌药物的敏感性。结果显示,山羊沙门氏菌的平均带菌率为54.59%。药敏试验结果显示,分离株均对丁胺卡那敏感,而对其余14种抗菌药物呈不同程度的耐药,多重耐药菌株占88%,其中耐2~7种药物的占52%,耐9~13种药物的占36%。结果表明,四川省部分地区山羊的沙门氏菌健康带菌率较高,且多重耐药性普遍,其公共卫生意义值得进一步研究。     

Genomic Description of Antibiotic Resistance in Escherichia coli and Enterococci Isolates from Healthy Lusitano Horses

Antibiotic resistance is a global problem, and it is known that commensal bacteria can act as reservoir of antibiotic resistance genes of clinical importance. The aim of the present study was to determine the antibiotic resistance phenotype and mechanisms implicated in resistance of Escherichia coli and Enterococcus spp. isolates collected from fecal samples of 90 Lusitano horses from Portugal. Sixteen of the 71 E. coli isolates (22.5%) recovered showed resistance to at least one of the antibiotics tested. The number of E. coli isolates resistant to streptomycin, tetracycline, chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole, and gentamicin was 9, 7, 6, 3, 2, and 1, respectively. The bla_TEM-1 and bla_OXA-1 genes were detected in ampicillin-resistant isolates and the sul2 and dfrA1 genes in trimethoprim-sulfamethoxazole-resistant, while the aac(3)-I, floR and tet(A) were found in the gentamicin, chloramphenicol and tetracycline-resistant isolates, respectively. Twenty-two of the 71 (31%) recovered enterococci showed antibiotic resistance for at least one of the tested antibiotics, and resistant isolates were identified as Enterococcus faecium (n = 14), E. faecalis (n = 3), E. hirae (n = 2), and Enterococcus spp. (n = 3). The erm(B) and erm(C) genes were identified in erythromycin-resistant enterococci and the tet(M) and/or tet(L) genes in tetracycline-resistant isolates. The slight prevalence of antibiotic resistance among commensal bacteria of healthy Lusitano horses can improve the treatment of upcoming infections in these horses because these microorganisms can be considered as antimicrobial indicator bacteria.     

动物源性沙门氏菌的耐药性分析及氟苯尼考类耐药基因的鉴定

为了解动物沙门氏菌的流行情况和药物敏感性及氟苯尼考耐药株的耐药基因分布,本试验对临床上疑似患沙门氏菌病的病料进行病原分离和细菌的多重PCR鉴定;采用K-B法测定分离株对23种抗菌药物的敏感性;选择氟苯尼考耐药菌株扩增floR、fexA、fexB、cfr和pexA基因。结果显示,共鉴定出61株沙门氏菌,其中肠炎沙门氏菌10株,鸡白痢沙门氏菌12株,鼠伤寒沙门氏菌39株。所有菌株对青霉素、红霉素、万古霉素耐药,90.16%对6种及6种以上抗菌药耐药。floR基因广泛存在于鼠伤寒沙门氏菌氟苯尼考耐药菌株中(8/12,66.67%),未发现其他耐药基因。研究结果表明鼠伤寒沙门氏菌是鹅源分离株中的优势血清型;floR基因主要介导沙门氏菌对氟苯尼考耐药性,但可能还存在其他机制。     

Resistance patterns and detection of aac(3)-IV gene in apramycin-resistant Escherichia coli isolated from farm animals and farm workers in northeastern of China

The aminoglycoside apramycin has been used widely in animal production in China since 1999. This study was aimed to investigate the resistance pattern of apramycin-resistant Escherichia coli isolated from farm animals and farm workers in northeastern of China during 2004–2007 and to determine whether resistance to apramycin was mediated by plasmid containing the aac(3)-IV gene and the mode for the transfer of genetic information between bacteria of farm animals and farm workers. Thirty six E. coli isolates of swine, chicken, and human origins, chosen randomly from 318 apramycin-resistant E. coli isolates of six farms in northeastern of China during 2004–2007, were multi-resistant and carried the aac(3)-IV gene encoding resistance to apramycin. Conjugation experiments demonstrated that in all 36 cases, the gene encoding resistance to apramycin was borne on a mobilisable plasmid. Homology analysis of the cloned aac(3)-IV gene with the sequence (accession no. X01385) in GenBank showed 99.3% identity at a nucleotide level, but only with a deletion of guanosine in position 813 of the gene in all 36 cases. The results indicted that resistance to apramycin in these isolates was closely related to aac(3)-IV gene. Therefore, the multi-resistance of E. coli could complicate therapeutic practices for enteric infections in both farm animals and human.     

Characterization of shiga toxin-producing Escherichia coli strains isolated from calves in Poland

Fecal samples from 67 3–5-months-old calves with diarrhea were screened for the presence of shiga toxin-producing Escherichia coli (STEC). Several accessory virulence factors genes were also tested. Among 192 E.coli isolates tested, 15 (7.6%) were found to harbour the shiga toxin 1 or 2 (stx1 or stx2) genes. The stx2-carrying samples were further subtyped by PCR for the stx2c, stx2d, and stx2e toxin variants. It was shown that stx2-positive bacteria mainly possessed the stx2c shiga toxin type gene. The enterohemolysin (hlyA) and intimin (eae) genes were found in seven (46.7%) STEC strains whereas the cytotoxic necrotizin factor 1 and 2 or the P fimbrial genes were detected in two isolates only. This study confirmed that calves are a reservoir of STEC strains (with all pathogenicity genes) that may be virulent for humans.     

Prevalence of antimicrobial resistance in enteric Escherichia coli from domestic pets and assessment of associated risk markers using a generalized linear mixed model

Antimicrobial resistance (AMR) is a growing global public health problem, which is caused by the use of antimicrobials in both human and animal medical practice. The objectives of the present cross-sectional study were as follows: (1) to determine the prevalence of resistance in Escherichia coli isolated from the feces of pets from the Porto region of Portugal against 19 antimicrobial agents and (2) to assess the individual, clinical and environmental characteristics associated with each pet as risk markers for the AMR of the E. coli isolates.     

川西北牦牛肉中产志贺毒素大肠杆菌的分离鉴定及stx2基因的序列分析

为了解中国川西北牦牛肉中产志贺毒素大肠杆菌(STEC)的携带情况及stx2的亚型和特征,试验将采集的204份川西北牦牛肉样品(各25g)增菌培养后,每份挑取5个可疑菌落,采用stx1、stx2双重PCR方法检测STEC,对分离株中的stx2分型并克隆测定stx2编码区序列。结果显示,在204份样品中分离出8株STEC,平均分离率为3.9%(8/204);存在4个不同的O血清型,分别为O38(4)、O50(1)、O74(2)、O150(1);在6株含有stx2的菌株中,其中有2株为stx2a型、4株为stx2c型。结果表明牦牛源分离株氨基酸序列与人源和牛源菌株同源性较高;由stx2A、B亚基的氨基酸序列系统进化树可知,牦牛源分离株与人源、牛源菌株聚为一支,表明它们之间遗传距离相对较近,牦牛源stx2各自分布在自己的小分支中,表明牦牛源STEC stx2与人源和牛源stx2相比,尽管亲缘关系较近,但仍存在一定程度的差异。     

Preliminary findings of Salmonella spp. in captive green iguanas (Iguana iguana) and their environment

Captive reptiles are routinely identified as reservoirs of Salmonella spp. and reports of reptile-associated salmonellosis are increasing. Unfortunately, little is known about the epidemiology of Salmonella spp. and green iguanas. We did a limited survey of a green-iguana farm in El Salvador to identify sources of Salmonella spp. in green iguanas and their environment. A limited number of samples for microbiological culture were collected from iguanas (adult, hatchling, and embryos) and their environment (food, water, soil, shelter, insects, and wild-caught lizards). Salmonella spp. was isolated from the intestine of both adult (3/20) and hatchling iguanas (8/20). There was no evidence of Salmonella spp. in the reproductive tracts of female iguanas (0/10). Salmonella spp. was isolated from the surface of 40% (7/16) of the egg surfaces tested. Salmonella spp. was not identified from the externalized yolk-sac of the iguana embryos tested. Soil samples from a breeding pen and a nest were both positive for Salmonella spp. Eight different Salmonella spp. serotypes were identified in this survey. These results suggest that horizontal transmission of Salmonella spp. is a potential source of exposure to hatchling iguanas at this facility.     

Occurrence of non-sorbitol fermenting, verocytotoxin-lacking Escherichia coli O157 on cattle farms

Escherichia coli O157 is often associated with hemorrhagic colitis and the hemolytic uremic syndrome (HUS). The verocytotoxins are considered to be the major virulence determinants. However, vt-negative E. coli O157 were recently isolated from patients with HUS. Several transmission routes to humans are described, but cattle feces are the primary source from which both the food supply and the environment become contaminated with E. coli O157.In a prevalence study performed on dairy, beef, mixed dairy/beef and veal farms in the summer of 2007, vt-negative isolates were detected on 11.8% (8/68) of the positive farms. From these eight farms, a total of 43 sorbitol-negative E. coli O157:H7 were collected. On five farms, only strains negative for the vt genes were present whereas both vt-negative and vt-positive strains could be detected on three other farms. Further characterization revealed that all isolates carried the eaeA and hlyA genes. Pulsed-field gel electrophoresis (PFGE) of all isolates resulted in nine different PFGE types and within the vt-negative strains, four different genotypes were identified, indicating that certain genetic clones are widespread over the cattle population.     

Verocytotoxins (Shiga-like toxins) produced by Escherichia coli: a minireview of their classification, clinical presentations and management of a heterogeneous family of cytotoxins

Bacterial virulence usually requires the interaction of multiple factors in order to cause disease. The enterotoxins produced by certain strains of bacteria are proteins which vary in their mode of action, but do fall into two general groups; the cytotoxic and the cytotonic enterotoxins. While cytotoxic enterotoxins typically kill eucaryotic cells (eg. by inhibiting protein synthesis), cytotonic enterotoxins derange cell metabolism in specific ways (eg. by elevating cyclic nucleotide levels). Some strains of Escherichia coli produce protein toxins that are biologically, structurally and antigenically related to a cytotoxin (Shiga toxin) (ShT) produced by Shigella dysenteriae type 1. Although this group of related, but not necessarily identical toxins have been referred to as Vero cell toxins or Verocytotoxins (VTs), the term Shiga-like toxins (SLTs) has been widely accepted. ShT and SLTs have been implicated as a cause of diarrhoea as well as haemorrhagic colitis (HC) and haemolytic uremic syndrome (HUS) in humans, whilst SLTs have been implicated as causal agents of oedema disease and HC in weaner pigs and calves, respectively. While S. dysenteriae is an invasive organism, the SLT-producing strains of E. coli have not been reported to be invasive, but cause diarrhoea that may contain blood and mucus. Thus, SLTs can be considered an important "new" type of enterotoxins whose role in the pathogenesis of diarrhoea, HC and HUS is beginning to emerge, not only in certain geographical settings, but worldwide. This mini review focuses on this family of SLTs, because of recent advances which have been made towards their detection, nomenclature, pathogenesis and possible management of their clinical presentations.