Influence of lincomycin-spectinomycin treatment on the outcome of Enterococcus cecorum infection and on the cecal microbiota in broilers

Background Enterococcus cecorum (EC) is one of the main reasons for skeletal disease in meat type chickens. Intervention strategies are still rare and focus mainly on early antibiotic treatment of the disease, although there are no data available concerning the effectivity of this procedure. The present study aimed to investigate the effectivity of early lincomycin-spectinomycin treatment during the first week of life after EC-infection. Furthermore, the impact of lincomycin-spectinomycin treatment and EC infection on the development of cecal microbiota was investigated. Methods A total of 383 day-old broiler chicks were randomly assigned to four groups (non-infected and non-treated, non-infected and treated, EC-infected and non-treated, and EC-infected and treated). The EC-infected groups were inoculated orally with an EC suspension at the day of arrival and at study day 3. The treatment groups were treated with lincomycin-spectinomycin via the drinking water for six consecutive days, starting two hours after the first inoculation. Necropsy of 20 chickens per group was performed at study days 7, 14, 21, and 42. Bacteriological examination via culture and real-time PCR was performed to detect EC in different extraintestinal organs. Cecal samples of nine chickens per group and necropsy day were analyzed to characterize the composition of the cecal microbiota. Results No clinical signs or pathologic lesions were found at necropsy, and EC was not detected in extraintestinal organs of the EC-infected and treated birds. Lincomycin-spectinomycin promoted the growth of the bacterial genus Escherichia/Shigella and reduced the amount of potentially beneficial Lactobacillus spp. in the ceca regardless of EC-infection. Unexpectedly, the highest abundances of the genus Enterococcus were found directly after ending antibiotic treatment in both treatment groups, suggesting the growth of resistant enterococcal species. EC was not detected among the most abundant members of the genus Enterococcus. Oral EC-infection at the first day of life did not influence the development of cecal microbiota in the present study. Conclusions Lincomycin-spectinomycin treatment during the first week of life can prevent the EC-associated disease in broiler type chickens and has a direct impact on the development of the cecal microbiota. The low abundance of EC in the ceca of infected chickens underlines the pathogenic nature of the disease-causing EC strains. Further research on alternative prevention and intervention strategies is needed with regard to current efforts on reducing the use of antibiotics in livestock animals.

Characterization and antimicrobial profile of enterococcal species from various clinical samples in a tertiary care centre

have long been recognised as low virulence bacteria occurring as commensals in the human intestine. However in the last two decades they emerged as one of the leading causes of nosocomial infections with the development of resistance to antibiotics. So appropriate identification and characterization and antimicrobial susceptibility testing of Enterococcal species is necessary for management and prevention of these infections.150 isolates of Enterococcal species were obtained from various clinical samples. Characterisation was done by standard Microbiological methods and antibiotic susceptibility testing was done by Kirby- Bauer disc diffusion method and Vancomycin MIC tested by E- test.Out of 150 isolates from various clinical samples like urine 93(62%), pus 45(30%), blood 7(4.6%) and other body fluids 5(3%), 131(87.3%) was the predominant isolate followed by 14(9.3%), 2(1.3%), 2(1.3%) and 1(0.6%). All isolates were sensitive to Vancomycin, Teicoplanin and Linezolid. Sensitivity to High level Gentamicin was 92%. Rate of resistance to Penicillin 150(100%), Tetracycline 95(63.3%), Ciprofloxacin 103(68.6%) and Ampicillin 67(44.6%).Even though no Vancomycin resistant strains were isolated from our study, there is incidence of Vancomycin resistant are emerging as potent pathogen. So methods for characterization, antimicrobial susceptibility testing and MIC of Vancomycin should be done routinely for Enterococcal species.

Clinical Impact of the Expanded BioFire Blood Culture Identification 2 Panel in a U.S. Children’s Hospital

The BioFire BCID2 panel is an accurate panel that is equivalent to the BioFire BCID panel compared to standard culture. The BioFire BCID2 panel offers several advantages over the BioFire BCID panel, including enterococcal species identification, Gram-negative resistance gene detection, Salmonella identification, and the added mecA / mecC and MREJ target for better Staphylococcus aureus and coagulase-negative Staphylococcus (CoNS) differentiation.

Enterococcal Species Associated with Slovak Raw Goat Milk, Their Safety and Susceptibility to Lantibiotics and Durancin ED26E/7

Goat milk has become a popular item of human consumption due to its originality. Enterococci are ubiquitous bacteria, and they can also be found in traditional dairy products. This study focuses on the safety of enterococci from Slovak raw goat milk and on their susceptibility to lantibiotic bacteriocins and durancin ED26E/7, which has not previously been studied. Biofilm formation ability in enterococci, virulence factor genes, enzyme production and antibiotic profile were investigated. Samples of raw goat milk (53) were collected from 283 goats in Slovakia. MALDI-TOF mass spectrometry identified three enterococcal species: Enterococcus faecium, E. hirae and E. mundtii, with dominant occurrence of the species E. faecium. Low-grade biofilm formation ability (0.1 ≤ A570 < 1.0) was found in four strains of E. faecium.Gelatinase, hyaluronidase, aggregation substance and enterococcal surface protein genes were absent in these enterococci. Gene efaAfm (adhesin) was detected in five E. faecium strains. However, it was not detected in biofilm-forming strains. Enterococci detected in Slovak raw goat milk were found not to have pathogenic potential; four strains even produced high amounts of useful β-galactosidase. The strains were susceptible to lantibiotic bacteriocin treatment and to durancin ED26E/7 as well, which represents original information in dairy production.

DIAGNOSTIC ACCURACY OF IN-HOUSE BIOCHEMICAL TESTING FOR IDENTIFICATION OF ENTEROCOCCUS SPECIES ISOLATED FROM VARIOUS CLINICAL SPECIMENSAGAINST VITEK 2 SYSTEM

Objective: To determine the diagnostic accuracy of in-house biochemical testing for identification of enterococcus species isolated from various clinical specimens against gold standard i.e., automated Vitek 2 system. This study also includes the antimicrobial susceptibility testing of enterococci against various antimicrobials. Study Design: Cross-sectional comparative study. Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi,from Apr 2017 to Mar 2018. Methodology: A total of 218 isolates from various clinical specimens suspected to be Enterococcus spp. werechecked by in-house biochemical testing including bile esculin, 6.5% NaCl and 1% arabinose and results werecompared with Vitek 2 compact system. The frequencies were determined by both systems and antimicrobial susceptibility testing was performed by disk diffusion as per clinical and laboratory standards institute guidelines. Results: Comparing the results of in-house testing with gold standard i.e., Vitek 2 system, the statistical data was calculated. Sensitivity turned out to be 100%, Specificity was found to be 68.75%. Positive and negative predictive values were 97.58% and 100% respectively. Accuracy turned out to be 97.71%. Conclusion: The in-house biochemical testing can be quite a useful method for identification of enterococci inresource-limited settings. However, it requires overight incubation and cannot identify other enterococcal species and non-enterococcal species. Vitek 2 is an automated system that is easy-to-handle, provides a rapid and reasonably accurate identification of enterococci alongwith accurate AST results. Enterococcal isolates from various clinical specimens in our setup showed least resistance to linezolid, followed by teicoplanin and vancomycin. Nitrofurantoin and.............

Identification of Clinically Relevant Streptococcus and Enterococcus Species Based on Biochemical Methods and 16S rRNA, sodA, tuf, rpoB, and recA Gene Sequencing

Streptococci and enterococci are significant opportunistic pathogens in epidemiology and infectious medicine. High genetic and taxonomic similarities and several reclassifications within genera are the most challenging in species identification. The aim of this study was to identify Streptococcus and Enterococcus species using genetic and phenotypic methods and to determine the most discriminatory identification method. Thirty strains recovered from clinical samples representing 15 streptococcal species, five enterococcal species, and four nonstreptococcal species were subjected to bacterial identification by the Vitek® 2 system and Sanger-based sequencing methods targeting the 16S rRNA, sodA, tuf, rpoB, and recA genes. Phenotypic methods allowed the identification of 10 streptococcal strains, five enterococcal strains, and four nonstreptococcal strains (Leuconostoc, Granulicatella, and Globicatella genera). The combination of sequencing methods allowed the identification of 21 streptococcal strains, five enterococcal strains, and four nonstreptococcal strains. The 16S rRNA and rpoB genes had the highest identification potential. Only a combination of several molecular methods was sufficient for unambiguous confirmation of species identity. This study will be useful for comparison of several identification methods, both those used as a first choice in routine microbiology and those used for final confirmation.

Vancomycin-Nonsusceptible Enterococci Mediated by vanC at a Large Children’s Hospital: Prevalence, Susceptibility, and Impact on Care of Enterococcal Bacteremia

Enterococcus gallinarum and casseliflavus have inherent vancomycin resistance and, though known as pathogens, have not been well characterized in pediatric patients. We identified a significant prevalence of these enterococcal species among immunocompromised patients at a large pediatric institution and describe the impact on patient care, antibiotic stewardship, and infection control.

Prevalence, antibiotic susceptibility and virulence factors of Enterococcus species in racing pigeons (Columba livia f. domestica)

Background This study was aimed to investigate the intestinal microbiota in racing pigeons with regard to Enterococcus species distribution, virulence factors and antibiotic susceptibility. Three methods (API, Multiplex sodA-PCR, 16S rRNA sequencing) were compared for Enterococcus species identification. Cloacal samples from 179 apparently healthy pigeons of 13 different flocks were tested. Results Multiplex sodA-PCR and 16S rRNA gene sequencing showed almost perfect agreement in Enterococcus species identification. Isolates were identified as Enterococcus columbae (34.5%), Enterococcus hirae (20.7%), Enterococcus faecalis (11.7%), Enterococcus faecium (11.7%), Enterococcus gallinarum (9%), Enterococcus mundtii (4.8%), Enterococcus casseliflavus (3.4%), Enterococcus cecorum (2.1%), Enterococcus durans (2.1%). More Enterococcus species were found after the race season than before. The study showed differences between Enterococcus species in relation to 68.8% (22/32) biochemical parameters. Six out of seven virulence genes were detected: gelE (43.5%), asa1 (42.1%), efaA (30.3%), ace (30.3%), cylA (27.6%), and esp (9%). None of the isolates harboured hyl gene. Overall 15.2% of Enterococcus isolates produced gelatinase, but 66.7% gelE genes were silent. Enterococcus faecalis showed the most often efaA, ace and gelatinase activity than other enterococcal species. Nearly all isolates (93.1%) were resistant to at least one antibiotic. The most frequent resistance was to enrofloxacin (80%), doxycycline with teicoplanin (73.1%), erythromycin (49.7%). The study revealed significant differences between some enterococcal species in the antibiotic susceptibility to different antibiotics. Enterococcus columbae and E. cecorum showed significantly more frequent resistance to chloramphenicol than other enterococci. The presence of VRE (19.3%), HLGR (2.8%) and no LRE were found. Overall 30.3% of isolates were positive for vancomycin resistance genes, where vanC1 (E. gallinarum), vanC2-C3 (E. hirae, E. casseliflavus), vanB (E. columbae) predominated. Conclusions We conclude, that intestinal microbiota in racing pigeons is composed by 9 different Enterococcus species. Given that racing pigeons are kept in close contact with humans and backyard animals, combined with their long-distance flight abilities, they can serve as potential source of virulent and antibiotic resistant Enterococcus spp. in the environment.

Development of a reference data set for assigning Streptococcus and Enterococcus species based on next generation sequencing of the 16S–23S rRNA region

Background Many members of Streptococcus and Enterococcus genera are clinically relevant opportunistic pathogens warranting accurate and rapid identification for targeted therapy. Currently, the developed method based on next generation sequencing (NGS) of the 16S–23S rRNA region proved to be a rapid, reliable and precise approach for species identification directly from polymicrobial and challenging clinical samples. The introduction of this new method to routine diagnostics is hindered by a lack of the reference sequences for the 16S–23S rRNA region for many bacterial species. The aim of this study was to develop a careful assignment for streptococcal and enterococcal species based on NGS of the 16S–23S rRNA region. Methods Thirty two strains recovered from clinical samples and 19 reference strains representing 42 streptococcal species and nine enterococcal species were subjected to bacterial identification by four Sanger-based sequencing methods targeting the genes encoding (i) 16S rRNA, (ii) sodA, (iii) tuf and (iv) rpoB; and NGS of the 16S–23S rRNA region. Results This study allowed obtainment and deposition of reference sequences of the 16S–23S rRNA region for 15 streptococcal and 3 enterococcal species followed by enrichment for 27 and 6 species, respectively, for which reference sequences were available in the databases. For Streptococcus, NGS of the 16S–23S rRNA region was as discriminative as Sanger sequencing of the tuf and rpoB genes allowing for an unambiguous identification of 93% of analyzed species. For Enterococcus, sodA, tuf and rpoB genes sequencing allowed for identification of all species, while the NGS-based method did not allow for identification of only one enterococcal species. For both genera, the sequence analysis of the 16S rRNA gene was endowed with a low identification potential and was inferior to that of other tested identification methods. Moreover, in case of phylogenetically related species the sequence analysis of only the intergenic spacer region was not sufficient enough to precisely identify Streptococcus strains at the species level. Conclusions Based on the developed reference dataset, clinically relevant streptococcal and enterococcal species can now be reliably identified by 16S–23S rRNA sequences in samples. This study will be useful for introduction of a novel diagnostic tool, NGS of the 16S–23S rRNA region, which undoubtedly is an improvement for reliable culture-independent species identification directly from polymicrobially constituted clinical samples.