Clonal Populations of Thermotolerant Enterobacteriaceae in Recreational Water and Their Potential Interference with Fecal Escherichia coli Counts

Bacterial strains were isolated from beach water samples using the original Environmental Protection Agency method for Escherichia coli enumeration and analyzed by pulsed-field gel electrophoresis (PFGE). Identical PFGE patterns were found for numerous isolates from 4 of the 9 days sampled, suggesting environmental replication. 16S rRNA gene sequencing, API 20E biochemical testing, and the absence of β-glucuronidase activity revealed that these clonal isolates were Klebsiella, Citrobacter, and Enterobacter spp. In contrast, 82% of the nonclonal isolates from water samples were confirmed to be E. coli, and 16% were identified as other fecal coliforms. These nonclonal isolates produced a diverse range of PFGE patterns similar to those of isolates obtained directly from untreated sewage and gull droppings. β-Glucuronidase activity was critical in distinguishing E. coli from other fecal coliforms, particularly for the clonal isolates. These findings demonstrate that E. coli is a better indicator of fecal pollution than fecal coliforms, which may replicate in the environment and falsely elevate indicator organism levels.     

Longitudinal Characterization of Resistant Escherichia coli in Fecal Deposits from Cattle Fed Subtherapeutic Levels of Antimicrobials

Model fecal deposits from cattle fed or not fed antimicrobial growth promoters were examined over 175 days in the field for growth and persistence of total Escherichia coli and numbers and proportions of ampicillin-resistant (Amp^r) and tetracycline-resistant (Tet^r) E. coli. In addition, genotypic diversity and the frequency of genetic determinants encoded by Amp^r and Tet^r E. coli were investigated. Cattle were fed diets containing chlortetracycline (44 ppm; A44 treatment group), chlortetracycline plus sulfamethazine (both at 44 ppm; AS700 treatment group), or no antibiotics (control). Fecal deposits were sampled 12 times over 175 days. Numbers of Tet^r E. coli in A44 and AS700 deposits were higher (P < 0.001) than those of controls and represented up to 35.6% and 20.2% of total E. coli, respectively. A time-by-treatment interaction (P < 0.001) was observed for the numbers of Tet^r and Amp^r E. coli. Except for Amp^r E. coli in control deposits, all E. coli numbers increased (P < 0.001) in deposits up to day 56. Even after 175 days, high Tet^r E. coli numbers were detected in A44 and AS700 deposits [5.9 log₁₀ CFU (g dry matter)⁻¹ and 5.4 log₁₀ CFU (g dry matter)⁻¹, respectively]. E. coli genotypes, as determined by pulsed-field gel electrophoresis, were diverse and were influenced by the antimicrobial growth promoter and the sampling time. Of the determinants screened, bla_TEM1, tetA, tetB, tetC, sul1, and sul2 were frequently detected. Occurrence of determinants was influenced by the feeding of antimicrobials. Fecal deposits remain a source of resistant E. coli even after a considerable period of environmental exposure.In North America antibiotics are widely used in beef cattle production as prophylactics or antimicrobial growth promoters (AGP). Used in this manner, antibiotics are generally administered in the diet either at times of high disease risk or on a continuous basis to improve feed efficiency. Employment of AGP in this manner may increase the prevalence of commensal antimicrobial-resistant (AR) bacteria (1, 41).There is evidence that resistant bacteria can be transferred from livestock to humans (5, 39, 55), and consequently the use of AGP has raised public health concerns. While the modes of transmission of AR bacteria and gene determinants are complex (33), survivability in agriculture-related matrices may be a critical factor in their dissemination (54). Most research on the persistence of AR bacteria in livestock waste has focused on large-scale management systems, such as land-applied manure (20, 44) or storage lagoons and pits (28, 49). In some instances, these systems have been shown to decrease the survival of select bacteria and the presence of AR genes (35, 51). Nevertheless, there is limited knowledge of the fate of AR bacteria or resistance determinants in feces shed from individuals, such as the fecal deposits that are formed in feedlot pens on intensively managed farms. Previous work has shown that fecal deposits provide an environment that is conducive to the growth and survival of pathogenic and commensal Escherichia coli (4, 48, 57). Because cattle fed AGP excrete antimicrobial residues in their feces (3), the residues may exert a selection pressure on bacteria after deposition of feces and potentially confer a growth advantage to resistant bacteria.The objective of the current study was to investigate both the longitudinal phenotypic and genotypic ecologies of AR E. coli in fecal deposits arising from groups of cattle administered either no AGP or one of two tetracycline-based AGP that are commonly used in the North American industry. We focused on ampicillin-resistant (Amp^r) and tetracycline-resistant (Tet^r) E. coli because both AGP treatments contained tetracycline, and we have previously found a link between the use of tetracycline-based AGP and Amp^r E. coli (1). We hypothesized that resistant E. coli would be more numerous in fecal deposits from animals previously fed AGP.     

Fecal Carriage of ESBL-Producing E. coli and K. pneumoniae in Children in Guinea-Bissau: A Hospital-Based Cross-Sectional Study

Background

In recent years, the world has seen a surge in extended-spectrum β-lactamase (ESBL)-producing bacteria. However, data on the dissemination of ESBL-producing Enterobacteriaceae in the community from systematically enrolled study subjects in Africa remains limited. To determine the prevalence, phenotypic resistance patterns and genetic characteristics of ESBL-producing E. coli and K. pneumoniae in fecal carriage and to analyze associated risk factors in children attending a pediatric emergency department in Guinea-Bissau.

Methodology/Principal Findings

From June to September 2010, children <5 years of age with fever or tachycardia attending a pediatric emergency ward during the day was screened for ESBL carriage in feces. Socio-demographic and health seeking behavior data was collected. Antibiotic susceptibility was tested with VITEK2 and EUCAST disk diffusion method, molecular characterization of ESBL-encoding genes was performed with multiplex PCR and clonal relatedness was established by automated rep-PCR. Of 408 enrolled children 133 (32.6%) were ESBL carriers. In total, 83 E. coli and 91 K. pneumoniae ESBL-producing isolates were obtained. Nearly all isolates were multidrug-resistant. Co-resistance to ciprofloxacin, trimethoprim-sulfamethoxazole and aminoglycosides was common. Of the isolates, 38.5% were co-resistant to these classes plus extended-spectrum cephalosporins, which infers resistance to all easily available antibiotic agents for treatment of gram-negative sepsis in Guinea-Bissau. The predominant resistance-encoding gene subgroup was bla _CTX-M-1 and epidemiologic typing showed that the bacterial ESBL population was highly diverse both for E. coli and K. pneumoniae. Bed sharing with another child <5 years of age was a risk factor for ESBL carriage, indicating crowding as a potential risk factor for transmission of ESBL-producing bacteria.

Conclusions/Significance

Prevalence of ESBL-producing bacteria in this population was high and clonally diverse. This is alarming considering the limited diagnostic and treatment possibilities in Guinea-Bissau and other resource-poor countries.     

Longitudinal Study of Escherichia coli O157 in a Cattle Finishing Unit

In a longitudinal study in a Finnish cattle finishing unit we investigated excretion and sources of Escherichia coli O157 in bulls from postweaning until slaughter. Three groups of 31 to 42 calves were sampled in a calf transporter before they entered the farm and four to seven times at approximately monthly intervals at the farm. All calves sampled in the livestock transporter were negative for E. coli O157 on arrival, whereas positive animals were detected 1 day later. During the fattening period the E. coli O157 infection rate varied between 0 and 38.5%. The animals were also found to be shedding during the cold months. E. coli O157 was isolated from samples taken from water cups, floors, and feed passages. E. coli O157 was detected in 9.7 to 38.9% of the fecal samples taken at slaughter, while only two rumen samples and one carcass surface sample were found to be positive. E. coli O157 was isolated from barn surface samples more often when the enrichment time was 6 h than when the enrichment time was 24 h (P < 0.0001). Fecal samples taken at the abattoir had lower counts (≤0.4 MPN/g) than fecal samples at the farm (P < 0.05). E. coli O157 was isolated more often from 10-g fecal samples than from 1-g fecal samples (P < 0.0001). Most farm isolates belonged to one pulsed-field gel electrophoresis (PFGE) genotype (79.6%), and the rest belonged to closely related PFGE genotypes. In conclusion, this study indicated that the finishing unit rather than introduction of new cattle was the source of E. coli O157 at the farm and that E. coli O157 seemed to persist well on barn surfaces.     

粪便样品中大肠杆菌多态性分子研究

以粪便样品中分离到的大肠杆菌为研究对象,比较了3种不同方法在分离鉴定大肠杆菌过程中的应用。首先,通过传统方法从粪便样品中分离,筛选和确定了一批大肠杆菌疑似菌株,再用现代分子生物学方法对待鉴定的大肠杆菌疑似菌株,已知大肠杆菌MG1655以及几种其它细菌进行ARDRA(AmplifiedRibosomalDNARestrictionAnalysis)分析,最后利用ERIC-PCR技术在个体水平上分析菌株的多样性。结果表明,所有由传统方法确定的大肠杆菌疑似菌株和MG1655都属于同一ARDRA型,并与其它细菌的ARDRA条码型不同。这说明ARDRA分析得到的结果与传统分析方法的结果吻合,利用ARDRA分析可以区分大肠杆菌和其它肠道细菌。但是在本实验中ARDRA分析不能反映大肠杆菌中不同菌株之间的多样性,ERIC-PCR则可以区分它们。     

Age-Related Decline in Carriage of Ampicillin-Resistant Escherichia coli in Young Calves

The presence of ampicillin-resistant Escherichia coli (Amp^r E. coli) in the fecal flora of calves was monitored on a monthly basis in seven cohorts of calves. Calves were rapidly colonized by Amp^r E. coli, with peak prevalence in cohort calves observed in the 4 months after the calves were born. The prevalence of calves yielding Amp^r E. coli in cohorts consistently declined to low levels with increasing age of the calves (P < 0.001).     

Rectal Carriage of Enterohemorrhagic Escherichia coli O157 in Slaughtered Cattle

Escherichia coli O157:H7 is an important cause of diarrhea, hemorrhagic colitis, and potentially fatal human illness. Cattle are considered a primary reservoir of infection, and recent experimental evidence has indicated that the terminal rectum is the principal site of bacterial carriage. To test this finding in naturally colonized animals, intact rectum samples from 267 cattle in 24 separate lots were obtained immediately after slaughter, and fecal material and mucosal surfaces were cultured for E. coli O157 by direct and enrichment methods. Two locations, 1 and 15 cm proximal to the recto-anal junction, were tested. In total, 35 animals were positive for E. coli O157 at at least one of the sites and 232 animals were negative as determined by all tests. The frequency of isolation and the numbers of E. coli O157 cells were higher at the site closer to the recto-anal junction, confirming our previous experimental findings. We defined low- and high-level carriers as animals with E. coli O157 levels of <1 × 10³ CFU g⁻¹ or <1 × 10³ CFU ml⁻¹ and animals with E. coli O157 levels of ≥1 × 10³ CFU g⁻¹ or ≥1 × 10³ CFU ml⁻¹ in feces or tissues, respectively. High-level carriage was detected in 3.7% of the animals (95% confidence interval, 1.8 to 6.8%), and carriage on the mucosal surface of the terminal rectum was associated with high-level fecal excretion. In summary, our results support previous work demonstrating that the mucosal epithelium in the bovine terminal rectum is an important site for E. coli O157 carriage in cattle. The data also support the hypothesis that high-level fecal shedding (≥1 × 10³ CFU g of feces⁻¹) of enterohemorrhagic E. coli O157 results from colonization of this site.     

Longitudinal Study of Fecal Shedding of Escherichia coli O157:H7 in Feedlot Cattle: Predominance and Persistence of Specific Clonal Types despite Massive Cattle Population Turnover

Identification of the sources and methods of transmission of Escherichia coli O157:H7 in feedlot cattle may facilitate the development of on-farm control measures for this important food-borne pathogen. The prevalence of E. coli O157:H7 in fecal samples of commercial feedlot cattle in 20 feedlot pens between April and September 2000 was determined throughout the finishing feeding period prior to slaughter. Using immunomagnetic separation, E. coli O157:H7 was isolated from 636 of 4,790 (13%) fecal samples in this study, with highest prevalence earliest in the feeding period. No differences were observed in the fecal or water trough sediment prevalence values of E. coli O157:H7 in 10 pens supplied with chlorinated drinking water supplies compared with nonchlorinated water pens. Pulsed-field gel electrophoresis of XbaI-digested bacterial DNA of the 230 isolates obtained from eight of the pens revealed 56 unique restriction endonuclease digestion patterns (REDPs), although nearly 60% of the isolates belonged to a group of four closely related genetic subtypes that were present in each of the pens and throughout the sampling period. The other REDPs were typically transiently detected, often in single pens and on single sample dates, and in many cases were also closely related to the four predominant REDPs. The persistence and predominance of a few REDPs observed over the entire feeding period on this livestock operation highlight the importance of the farm environment, and not necessarily the incoming cattle, as a potential source or reservoir of E. coli O157:H7 on farms.     

Carriage of stx2a Differentiates Clinical and Bovine-Biased Strains of Escherichia coli O157

Background

Shiga toxin (Stx) are cardinal virulence factors of enterohemorrhagic E. coli O157:H7 (EHEC O157). The gene content and genomic insertion sites of Stx-associated bacteriophages differentiate clinical genotypes of EHEC O157 (CG, typical of clinical isolates) from bovine-biased genotypes (BBG, rarely identified among clinical isolates). This project was designed to identify bacteriophage-mediated differences that may affect the virulence of CG and BBG.

Methods

Stx-associated bacteriophage differences were identified by whole genome optical scans and characterized among >400 EHEC O157 clinical and cattle isolates by PCR.

Results

Optical restriction maps of BBG strains consistently differed from those of CG strains only in the chromosomal insertion sites of Stx2-associated bacteriophages. Multiplex PCRs (stx1, stx2a, and stx2c as well as Stx-associated bacteriophage - chromosomal insertion site junctions) revealed four CG and three BBG that accounted for >90% of isolates. All BBG contained stx2c and Stx2c-associated bacteriophage – sbcB junctions. All CG contained stx2a and Stx2a-associated bacteriophage junctions in wrbA or argW.

Conclusions

Presence or absence of stx2a (or another product encoded by the Stx2a-associated bacteriophage) is a parsimonious explanation for differential virulence of BBG and CG, as reflected in the distributions of these genotypes in humans and in the cattle reservoir.     

Identification of Fecal Escherichia coli from Humans and Animals by Ribotyping

Fecal pollution of water resources is an environmental problem of increasing importance. Identification of individual host sources of fecal Escherichia coli, such as humans, pets, production animals, and wild animals, is prerequisite to formulation of remediation plans. Ribotyping has been used to distinguish fecal E. coli of human origin from pooled fecal E. coli isolates of nonhuman origin. We have extended application of this technique to distinguishing fecal E. coli ribotype patterns from human and seven individual nonhuman hosts. Classification accuracy was best when the analysis was limited to three host sources. Application of this technique to identification of host sources of fecal coliforms in water could assist in formulation of pollution reduction plans.     

Longitudinal study of Escherichia coli O157 in a cattle finishing unit

In a longitudinal study in a Finnish cattle finishing unit we investigated excretion and sources of Escherichia coli O157 in bulls from postweaning until slaughter. Three groups of 31 to 42 calves were sampled in a calf transporter before they entered the farm and four to seven times at approximately monthly intervals at the farm. All calves sampled in the livestock transporter were negative for E. coli O157 on arrival, whereas positive animals were detected 1 day later. During the fattening period the E. coli O157 infection rate varied between 0 and 38.5%. The animals were also found to be shedding during the cold months. E. coli O157 was isolated from samples taken from water cups, floors, and feed passages. E. coli O157 was detected in 9.7 to 38.9% of the fecal samples taken at slaughter, while only two rumen samples and one carcass surface sample were found to be positive. E. coli O157 was isolated from barn surface samples more often when the enrichment time was 6 h than when the enrichment time was 24 h (P < 0.0001). Fecal samples taken at the abattoir had lower counts (< or = 0.4 MPN/g) than fecal samples at the farm (P < 0.05). E. coli O157 was isolated more often from 10-g fecal samples than from 1-g fecal samples (P < 0.0001). Most farm isolates belonged to one pulsed-field gel electrophoresis (PFGE) genotype (79.6%), and the rest belonged to closely related PFGE genotypes. In conclusion, this study indicated that the finishing unit rather than introduction of new cattle was the source of E. coli O157 at the farm and that E. coli O157 seemed to persist well on barn surfaces.     

Cefotaxime Resistant Escherichia coli Collected from a Healthy Volunteer; Characterisation and the Effect of Plasmid Loss

In this study 6 CTX-M positive E. coli isolates collected during a clinical study examining the effect of antibiotic use in a human trial were analysed. The aim of the study was to analyse these isolates and assess the effect of full or partial loss of plasmid genes on bacterial fitness and pathogenicity. A DNA array was utilised to assess resistance and virulence gene carriage. Plasmids were characterised by PCR-based replicon typing and addiction system multiplex PCR. A phenotypic array and insect virulence model were utilised to assess the effect of plasmid-loss in E. coli of a large multi-resistance plasmid. All six E. coli carrying bla_CTX-M-14 were detected from a single participant and were identical by pulse field gel electrophoresis and MLST. Plasmid profiling and arrays indicated absence of a large multi-drug resistance (MDR) F-replicon plasmid carrying blaTEM, aadA4, strA, strB, dfrA17/19, sul1, and tetB from one isolate. Although this isolate partially retained the plasmid it showed altered fitness characteristics e.g. inability to respire in presence of antiseptics, similar to a plasmid-cured strain. However, unlike the plasmid-cured or plasmid harbouring strains, the survival rate for Galleria mellonella infected by the former strain was approximately 5-times lower, indicating other possible changes accompanying partial plasmid loss. In conclusion, our results demonstrated that an apparently healthy individual can harbour bla_CTX-M-14E. coli strains. In one such strain, isolated from the same individual, partial absence of a large MDR plasmid resulted in altered fitness and virulence characteristics, which may have implications in the ability of this strain to infect and any subsequent treatment.     

Factors Associated to Prevalence and Incidence of Carbapenem-Resistant Enterobacteriaceae Fecal Carriage: A Cohort Study in a Mexican Tertiary Care Hospital

Background

Carbapenem-resistant Enterobacteriaceae (CRE) infections have emerged as a serious threat to health worldwide. They are associated with increased morbidity and mortality and are capable of silently colonizing the gastrointestinal tract. Because of this, there is great interest to characterize the epidemiology of CRE carriage and acquisition in healthcare facilities. The aim of this study was to determine the prevalence and factors associated with CRE fecal carriage (CRE-fc), and risk factors for incident cases.

Methods/Results

A cohort study was conducted at a tertiary care hospital from January 1^st to April 30^th, 2014 during a CRE outbreak. Weekly rectal swabs were performed in patients considered at risk until discharge. CRE-fc prevalence was 10.9% (CI 95% 7.7–14.7) among 330 patients. Treatment with carbapenems (OR 2.54, CI 95% 1.15–5.62); transfer from an institution (OR 2.16, CI 95% 1.02–4.59); multi-drug resistant infection within the previous six months (OR 2.81, CI 95% 1.47–5.36); intensive care unit admission (OR 0.42, CI 95% 0.20–0.88); hematologic malignancy (OR 4.02, CI 95% 1.88–8.06); invasive procedures (OR 2.18, CI 95% 1.10–4.32); and sharing a room with a known CRE carrier (OR 3.0, CI 95% 1.43–6.31) were independently associated factors for CRE-fc. Risk factors associated with CRE-fc incidence were determined for 87 patients initially negative and with subsequent screening; the incidence rate was 2.5 cases, per 1000 person-years (CI 95% 1.5–3.9). Independently associated risk factors were carbapenem treatment (HR 2.68, CI 95% 1.03–6.98), hematologic malignancy (HR 5.74, 95% CI 2.46–13.4) and a mean daily colonization pressure ≥10% (HR 5.03, IC 95% 1.77–14.28). OXA-48-like (OXA-232) and CTX-M-15 were the predominantly identified mechanisms of resistance.

Conclusions

We found an elevated incidence and prevalence of CRE-fc in our hospital. Hematologic patients need to be considered a population at risk, and antibiotic stewardship along with infection control programs need to be improved to avoid nosocomial spread.     

Longitudinal Emergence and Distribution of Escherichia coli O157 Genotypes in a Beef Feedlot

The purpose of this study was to describe the prevalence and longitudinal distribution of Escherichia coli O157 in feedlot cattle and the feedlot environment. Pen floors, water tanks, other cattle in the feedlot, feed, and bird feces were sampled for 2 weeks prior to entry of the study cattle. Twelve pens of study cattle were sampled twice weekly. At each sample time cattle feces, water from tanks in each pen, bunk feed, feed components, bird feces, and houseflies were collected. Bunk feed samples were collected before and after cattle had access to the feed. Overall, 28% of cattle fecal samples, 3.9% of bird fecal samples, 25% of water samples, 3.4% of housefly samples, 1.25% of bunk feed before calf access, and 3.25% of bunk feed samples after cattle had access to the feed were positive for E. coli O157. Genetic analysis of E. coli O157 isolates was done using pulsed-field gel electrophoresis (PFGE). PFGE types identified in sampling of the feedlot prior to calf entry were different than the majority of types identified following calf entry. A single strain type predominated in the samples collected after entry of the cattle. It was first identified 5 days after entry of the first pen of cattle and was subsequently identified in all pens. Data support that the incoming cattle introduced a new strain that became the predominant strain in the feedlot.     

Longitudinal emergence and distribution of Escherichia coli O157 genotypes in a beef feedlot

The purpose of this study was to describe the prevalence and longitudinal distribution of Escherichia coli O157 in feedlot cattle and the feedlot environment. Pen floors, water tanks, other cattle in the feedlot, feed, and bird feces were sampled for 2 weeks prior to entry of the study cattle. Twelve pens of study cattle were sampled twice weekly. At each sample time cattle feces, water from tanks in each pen, bunk feed, feed components, bird feces, and houseflies were collected. Bunk feed samples were collected before and after cattle had access to the feed. Overall, 28% of cattle fecal samples, 3.9% of bird fecal samples, 25% of water samples, 3.4% of housefly samples, 1.25% of bunk feed before calf access, and 3.25% of bunk feed samples after cattle had access to the feed were positive for E. coli O157. Genetic analysis of E. coli O157 isolates was done using pulsed-field gel electrophoresis (PFGE). PFGE types identified in sampling of the feedlot prior to calf entry were different than the majority of types identified following calf entry. A single strain type predominated in the samples collected after entry of the cattle. It was first identified 5 days after entry of the first pen of cattle and was subsequently identified in all pens. Data support that the incoming cattle introduced a new strain that became the predominant strain in the feedlot.