Impact of short-term storage on the quantity of extended-spectrum beta-lactamase–producing Escherichia coli in broiler litter under practical conditions

Applying broiler litter containing extended-spectrum beta-lactamase (ESBL)–producing Escherichia coli (E. coli) to arable land poses a potential risk for humans to get colonized by contact with contaminated soil or vegetables. Therefore, an inactivation of these bacteria before land application of litter is crucial. We performed 2 short-term litter storage trials (one in summer and winter, respectively), each covering a time span of 5 D to investigate the effectiveness of this method for inactivation of ESBL-producing E. coli in chicken litter. Surface and deep litter samples were taken from a stacked, ESBL-positive chicken litter heap in triplicates in close sampling intervals at the beginning and daily for the last 3 D of the experiments. Samples were analyzed quantitatively and qualitatively for ESBL-producing E. coli, total E. coli, and enterococci. Selected isolates were further characterized by whole-genome sequencing (WGS). In the depth of the heap ESBL-producing E. coli were detected quantitatively until 72 h and qualitatively until the end of the trial in winter. In summer detection was possible quantitatively up to 36 h and qualitatively until 72 h. For surface litter samples a qualitative detection of ESBL-producing E. coli was possible in all samples taken in both trials. In the deep samples a significant decrease in the bacterial counts of over 2 Log10 was observed for total E. coli in the winter and for total E. coli and enterococci in the summer. Genetic differences of the isolates analyzed by WGS did not correlate with survival advantage. In conclusion, short-term storage of chicken litter stacked in heaps is a useful tool for the reduction of bacterial counts including ESBL-producing E. coli. However, incomplete inactivation was observed at the surface of the heap and at low ambient temperatures. Therefore, an extension of the storage period in winter as well as turning of the heap to provide aerobic composting conditions should be considered if working and storage capacities are available on the farms

Typing Clostridium difficile strains based on tandem repeat sequences

Background: Genotyping of epidemic Clostridium difficile strains is necessary to track their emergence and spread. Portability of genotyping data is desirable to facilitate inter-laboratory comparisons and epidemiological studies. Results: This report presents results from a systematic screen for variation in repetitive DNA in the genome of C. difficile. We describe two tandem repeat loci, designated \u27TR6\u27 and \u27TR10\u27, which display extensive sequence variation that may be useful for sequence-based strain typing. Based on an investigation of 154 C. difficile isolates comprising 75 ribotypes, tandem repeat sequencing demonstrated excellent concordance with widely used PCR ribotyping and equal discriminatory power. Moreover, tandem repeat sequences enabled the reconstruction of the isolates\u27 largely clonal population structure and evolutionary history. Conclusion: We conclude that sequence analysis of the two repetitive loci introduced here may be highly useful for routine typing of C. difficile. Tandem repeat sequence typing resolves phylogenetic diversity to a level equivalent to PCR ribotypes. DNA sequences may be stored in databases accessible over the internet, obviating the need for the exchange of reference strains

Antibiotic resistance and molecular epidemiology of Staphylococcus aureus in Nigeria

<p>Abstract</p> <p>Background</p> <p><it>Staphylococcus aureus </it>is an important pathogen causing a wide range of infections in the hospital and community setting. In order to have adequate information for treatment of <it>S. aureus </it>infections, it is crucial to understand the trends in the antibiotic-resistance patterns. In addition, the occurrence and changes in types of <it>S. aureus</it>, clonal identities, and their geographic spread is essential for the establishment of adequate infection control programmes. In this study, 68 <it>S. aureus </it>isolates obtained from clinical and non-clinical sources in Nigeria between January and April 2009 were characterized using phenotypic and molecular methods.</p> <p>Results</p> <p>All the <it>S. aureus </it>isolates were susceptible to teicoplanin, vancomycin, phosphomycin, fusidic acid, rifampicin, daptomycin, mupirocin, linezolid and tigecycline. Sixteen percent of the isolates were resistant to oxacillin, while 55% and 72% of isolates were resistant to tetracycline and trimethoprim/sulphamethoxazole (cotrimoxazole), respectively (Table <tblr tid="T1">1</tblr>). There was excellent correlation between the broth microdilution assay and detection of antibiotic resistance genes by the multiplex PCR, in the determination of <it>S. aureus </it>resistance to erythromycin, gentamicin, methicillin and tetracycline. A total of 28 <it>spa </it>types were identified in the study, and the predominant <it>spa </it>type among the methicillin-susceptible <it>S. aureus </it>(MSSA) isolates was t084 (13 isolates). The t037-ST241-SCC<it>mec</it>III type was the only clone identified in Maiduguri (North-East Nigeria) while in South-West Nigeria, diversity among the MRSA isolates (t451-ST8-SCC<it>mec</it>V; t008-ST94-SCC<it>mec</it>IV; t002-ST5-SCC<it>mec</it>V; t064-ST8-SCC<it>mec</it>V) was observed. The toxin genes <it>seh </it>and <it>etd </it>were detected in isolates affiliated with clonal complexes CC1, CC80 and sequence type ST25, respectively. The proportion of PVL-positive isolates among MSSA was high (40%). Most of the PVL-positive MSSA isolates were obtained from wound infections and associated with clonal complexes CC1, CC30, CC121 and with sequence type ST152.</p> <tbl id="T1"> <title> <p>Table 1</p> </title> <caption> <p>Antibiotic resistance profile of <it>S. aureu</it><it>s </it>(MSSA and MRSA) from Nigeria</p> </caption> <tblbdy cols="4"> <r> <c> <p/> </c> <c cspan="3" ca="left"> <p><b>Number (%) of resistant isolates among</b>:</p> </c> </r> <r> <c ca="left"> <p><b>Antibiotic</b></p> </c> <c ca="left"> <p><b>MSSA</b></p> <p><b>(n = 57)</b></p> </c> <c ca="left"> <p><b>MRSA</b></p> <p><b>(n = 11)</b></p> </c> <c ca="left"> <p><b>Total</b></p> <p><b>(n = 68)</b></p> </c> </r> <r> <c cspan="4"> <hr/> </c> </r> <r> <c ca="left"> <p>Penicillin</p> </c> <c ca="left"> <p>49 (86)</p> </c> <c ca="left"> <p>11 (100)</p> </c> <c ca="left"> <p>60 (88.2)</p> </c> </r> <r> <c ca="left"> <p>Oxacillin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>11 (100)</p> </c> <c ca="left"> <p>11 (16.2)</p> </c> </r> <r> <c ca="left"> <p>Teicoplanin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> <r> <c ca="left"> <p>Vancomycin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> <r> <c ca="left"> <p>Gentamicin</p> </c> <c ca="left"> <p>1 (1.8)</p> </c> <c ca="left"> <p>9 (81.8)</p> </c> <c ca="left"> <p>10 (14.7)</p> </c> </r> <r> <c ca="left"> <p>Tetracycline</p> </c> <c ca="left"> <p>27 (47.4)</p> </c> <c ca="left"> <p>11 (100)</p> </c> <c ca="left"> <p>38 (55.9)</p> </c> </r> <r> <c ca="left"> <p>Ciprofloxacin</p> </c> <c ca="left"> <p>12 (21.1)</p> </c> <c ca="left"> <p>8 (72.7)</p> </c> <c ca="left"> <p>20 (29.4)</p> </c> </r> <r> <c ca="left"> <p>Moxifloxacin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>7 (63.6)</p> </c> <c ca="left"> <p>7 (10.3)</p> </c> </r> <r> <c ca="left"> <p>Trimethoprim/sulfamethoxazole</p> </c> <c ca="left"> <p>39 (68.4)</p> </c> <c ca="left"> <p>10 (90.9)</p> </c> <c ca="left"> <p>49 (72.1)</p> </c> </r> <r> <c ca="left"> <p>Phosphomycin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> <r> <c ca="left"> <p>Fusidic acid</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> <r> <c ca="left"> <p>Erythromycin</p> </c> <c ca="left"> <p>2 (3.5)</p> </c> <c ca="left"> <p>6 (54.5)</p> </c> <c ca="left"> <p>8 (11.8)</p> </c> </r> <r> <c ca="left"> <p>Clindamycin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>6 (54.5)</p> </c> <c ca="left"> <p>6 (8.8)</p> </c> </r> <r> <c ca="left"> <p>Rifampicin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> <r> <c ca="left"> <p>Daptomycin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> <r> <c ca="left"> <p>Mupirocin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> <r> <c ca="left"> <p>Linezolid</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> <r> <c ca="left"> <p>Tigecycline</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> </tblbdy> </tbl> <p>Conclusions</p> <p>The use of phenotypic and molecular methods provided useful information on antibiotic resistance and molecular diversity of <it>S. aureus </it>in Nigeria. The high proportion of PVL-positive MSSA isolates affiliated to various clonal complexes and detected in all the health institutions is a major concern, both as a source of severe infections and as a potential reservoir that could lead to the emergence of PVL-positive MRSA. This study presents the first baseline information on the nature of the antibiotic resistance genes from <it>S. aureus </it>isolates in Nigeria. There is the need to curtail the spread and establishment of MRSA and PVL-positive MSSA clones in Nigerian health care institutions.</p

Low airborne tenacity and spread of ESBL-/AmpC-producing Escherichia coli from fertilized soil by wind erosion

ESBL-/AmpC-producing Escherichia coli from organic fertilizers were previously detected on soil surfaces of arable land and might be emitted by wind erosion. To investigate this potential environmental transmission path, we exposed ESBL-/AmpC-positive chicken litter, incorporated in agricultural soils, to different wind velocities in a wind tunnel and took air samples for microbiological analysis. No data exist concerning the airborne tenacity of ESBL-/AmpC-producing E. coli. Therefore, we explored the tenacity of two ESBL/AmpC E. coli strains and E. coli K12 in aerosol chamber experiments at different environmental conditions. In the wind tunnel, ESBL/AmpC-producing E. coli were detected in none of the air samples (n = 66). Non-resistant E. coli were qualitatively detected in 40.7% of air samples taken at wind velocities exceeding 7.3 m s(-1). Significantly increased emission of total viable bacteria with increasing wind velocity was observed. In the aerosol chamber trials, recovery rates of airborne E. coli ranged from 0.003% to 2.8%, indicating a low airborne tenacity. Concluding, an emission of ESBL/AmpC E. coli by wind erosion in relevant concentrations appears unlikely because of the low concentration in chicken litter compared with non-resistant E. coli and their low airborne tenacity, proven in the aerosol chamber trials

Single-Nucleotide Polymorphism Genotyping Identifies a Locally Endemic Clone of Methicillin-Resistant Staphylococcus aureus

We developed, tested, and applied a TaqMan real-time PCR assay for interrogation of three single-nucleotide polymorphisms that differentiate a clade (termed ‘t003-X’) within the radiation of methicillin-resistant Staphylococcus aureus (MRSA) ST225. The TaqMan assay achieved 98% typeability and results were fully concordant with DNA sequencing. By applying this assay to 305 ST225 isolates from an international collection, we demonstrate that clade t003-X is endemic in a single acute-care hospital in Germany at least since 2006, where it has caused a substantial proportion of infections. The strain was also detected in another hospital located 16 kilometers away. Strikingly, however, clade t003-X was not found in 62 other hospitals throughout Germany nor among isolates from other countries, and, hence, displayed a very restricted geographical distribution. Consequently, our results show that SNP-typing may be useful to identify and track MRSA clones that are specific to individual healthcare institutions. In contrast, the spatial dissemination pattern observed here had not been resolved by other typing procedures, including multilocus sequence typing (MLST), spa typing, DNA macrorestriction, and multilocus variable-number tandem repeat analysis (MLVA)

Functional relationship of particulate matter (PM) emissions, animal species, and moisture content during manure application

Livestock manure is recycled to agricultural land as organic fertilizer. Due to the extensive usage of antibiotics in conventional animal farming, antibiotic-resistant bacteria are highly prevalent in feces and manure. The spread of wind-driven particulate matter (PM) with potentially associated harmful bacteria through manure application may pose a threat to environmental and human health. We studied whether PM was aerosolized during the application of solid and dried livestock manure and the functional relationship between PM release, manure dry matter content (DM), treatment and animal species. In parallel, manure and resulting PM were investigated for the survival of pathogenic and antibiotic-resistant bacterial species. The results showed that from manure with a higher DM smaller particles were generated and more PM was emitted. A positive correlation between manure DM and PM aerosolization rate was observed. There was a species-dependent critical dryness level (poultry: 60% DM, pig: 80% DM) where manure began to release PM into the environment. The maximum PM emission potentials were 1 and 3 kg t(-1) of applied poultry and pig manure, respectively. Dried manure and resulting PM contained strongly reduced amounts of investigated pathogenic and antibiotic-resistant microorganisms compared to fresh samples. An optimal manure DM regarding low PM emissions and reduced pathogen viability was defined from our results, which was 55-70% DM for poultry manure and 75-85% DM for pig manure. The novel findings of this study increase our detailed understanding and basic knowledge on manure PM emissions and enable optimization of manure management, aiming a manure DM that reduces PM emissions and pathogenic release into the environment

Molecular and microbiological report of a hospital outbreak of NDM-1-carrying Enterobacteriaceae in Mexico

Abstract Objectives To characterize the microbiological, molecular and epidemiological data of an outbreak of carbapenem-resistant Enterobacteriaceae (CRE) in a tertiary-care hospital in Mexico. Methods From September 2014 to July 2015, all CRE clinical isolates recovered during an outbreak in the Hospital Civil "Fray Antonio Alcalde" in Jalisco, Mexico were screened for antimicrobial susceptibility, carbapenemase production, carbapenemase-encoding genes, and plasmid profiles. Horizontal transfer of imipenem resistance; and clonal diversity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST); as well as biofilm production and the presence of 14 virulence genes were analyzed in selected isolates. Results Fifty-two carbapenem-resistant isolates corresponding to 5 species were detected, i.e., Klebsiella pneumoniae (n = 46), Enterobacter cloacae (n = 3), Escherichia coli (n = 1), Providencia rettgeri (n = 1) and Citrobacter freundii (n = 1) with carbapenemase encoding genes blaNDM-1 (n = 48), blaVIM (n = 3), blaIMP (n = 1) and blaKPC (n = 1) detected in these isolates. The blaNDM-1 gene was detected in plasmids from 130- to 170-kb in K. pneumoniae (n = 46); E. cloacae (n = 3), E. coli (n = 1) and P. rettgeri (n = 1). The transfer of plasmids harboring the blaNDM-1 gene was obtained in eight transconjugants. One plasmid restriction pattern was detected, with the blaNDM-1 identified in different restriction fragments. Predominant clone A of K. pneumoniae isolates archived 28/46 (60%) isolates and belongs to ST392. Besides, ST307, ST309, ST846, ST2399, and ST2400 were detected for K. pneumoniae; as well as E. cloacae ST182 and E. coli ST10. The fimA and uge genes were more likely to be identified in K. pneumoniae carbapenemsusceptible isolates (p =<0.001) and biofilm production was more liable to be observed in carbapenem-resistant isolates (p =<0.05). Conclusions Four Enterobacteriaceae species harboring the blaNDM-1 gene were detected in a nosocomial outbreak in Mexico; horizontal transfer and strain transmission were demonstrated for the blaNDM-1 gene. Given the variation in the size of the plasmid harboring blaNDM-1, complex rearrangements must also be occurring

Agricultural fertilization with poultry manure results in persistent environmental contamination with the pathogen Clostridioides difficile

During a field experiment applying broiler manure for fertilization of agricultural land, we detected viable Clostridioides (also known as Clostridium) difficile in broiler faeces, manure, dust and fertilized soil. A large diversity of toxigenic C. difficile isolates was recovered, including PCR ribotypes common from human disease. Genomic relatedness of C. difficile isolates from dust and from soil, recovered more than 2 years after fertilization, traced their origins to the specific chicken farm that had delivered the manure. We present evidence of long-term contamination of agricultural soil with manure-derived C. difficile and demonstrate the potential for airborne dispersal of C. difficile through dust emissions during manure application. Clostridioides genome sequences virtually identical to those from manure had been recovered from chicken meat and from human infections in previous studies, suggesting broiler-associated C. difficile are capable of zoonotic transmission

Particulate matter emissions during field application of poultry manure - The influence of moisture content and treatment

Along with industry and transportation, agriculture is one of the main sources of primary particulate matter (PM) emissions worldwide. Bioaerosol formation and PM release during livestock manure field application and the associated threats to environmental and human health are rarely investigated. In the temperate climate zone, field fertilization with manure seasonally contributes to local PM air pollution regularly twice per year (spring and autumn). Measurements in a wind tunnel, in the field and computational fluid dynamics (CFD) simulations were performed to analyze PM aerosolization during poultry manure application and the influence of manure moisture content and treatment. A positive correlation between manure dry matter content (DM) and PM release was observed. Therefore, treatments strongly increasing the DM of poultry manure should be avoided. However, high manure DM led to reduced microbial abundance and, therefore, to a lower risk of environmental pathogen dispersion. Considering the findings of PM and microbial measurements, the optimal poultry manure DM range for field fertilization was identified as 50–70%. Maximum PM10 concentrations of approx. 10 mg per m3 of air were measured during the spreading of dried manure (DM 80%), a concentration that is classified as strongly harmful. The modeling of PM aerosolization processes indicated a low health risk beyond a distance of 400 m from the manure application source. The detailed knowledge about PM aerosolization during manure field application was improved with this study, enabling manure management optimization for lower PM aerosolization and pathogenic release into the environment

Airborne bacterial emission fluxes from manure-fertilized agricultural soil

This is the first study to quantify the dependence on wind velocity of airborne bacterial emission fluxes from soil. It demonstrates that manure bacteria get aerosolized from fertilized soil more easily than soil bacteria, and it applies bacterial genomic sequencing for the first time to trace environmental faecal contamination back to its source in the chicken barn. We report quantitative, airborne emission fluxes of bacteria during and following the fertilization of agricultural soil with manure from broiler chickens. During the fertilization process, the concentration of airborne bacteria culturable on blood agar medium increased more than 600 000-fold, and 1 m3 of air carried 2.9 × 105 viable enterococci, i.e. indicators of faecal contamination which had been undetectable in background air samples. Trajectory modelling suggested that atmospheric residence times and dispersion pathways were dependent on the time of day at which fertilization was performed. Measurements in a wind tunnel indicated that airborne bacterial emission fluxes from freshly fertilized soil under local climatic conditions on average were 100-fold higher than a previous estimate of average emissions from land. Faecal bacteria collected from soil and dust up to seven weeks after fertilization could be traced to their origins in the poultry barn by genomic sequencing. Comparative analyses of 16S rRNA gene sequences from manure, soil and dust showed that manure bacteria got aerosolized preferably, likely due to their attachment to low-density manure particles. Our data show that fertilization with manure may cause substantial increases of bacterial emissions from agricultural land. After mechanical incorporation of manure into soil, however, the associated risk of airborne infection is low