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Efficacy of disinfectants against multidrug-resistant Enterobacter cloacae strains isolated from humans in a clinical settingGuenther, Phatchanok 07 December 2020 (has links)
Einführung: Enterobacter (E.) cloacae subsp. cloacae sind wichtige Humanpathogene, insbesondere bei stationär untergebrachten Patienten. Sie sind in der Lage, Medizinprodukte zu kontaminieren, und es wurde über nosokomiale Krankheitsausbrüche in Verbindung mit der Kolonisation von chirurgischen Utensilien berichtet. Es ist daher wichtig, die Wirksamkeit und Effizienz von Desinfektionsmitteln gegenüber dieser Bakterienart zu bestimmen.
Ziele: Die aktuelle Studie wurde durchgeführt, um nachzuweisen, ob Peressigsäure, Ethanol, Benzalkoniumchlorid und Natriumhypochlorit, welche weit verbreitet in kommerziellen Desinfektionsmitteln enthalten sind, eine ausreichende Wirksamkeit gegen multiresistente, von Patienten im Krankenhaus isolierte, E. cloacae aufweisen.
Material und Methoden: Sechs multiresistente E. cloacae Isolate, die von Patienten in einem klinischen Umfeld gewonnen wurden, wurden getestet und mit dem E. cloacae Typstamm verglichen. Die Studien wurden in vitro mit Peressigsäure, Ethanol, Benzalkoniumchlorid und Natriumhypochlorit nach den Richtlinien der Desinfektionsmittel-Kommission des Verbundes für Angewandte Hygiene e.V. durchgeführt. Die Tests umfassten qualitative und quantitative Suspensionstests zur Bestimmung der bakteriziden Wirkung, den sogenannten Keimträgertest und die Bestimmung der minimalen Hemmkonzentrationen.
Ergebnisse: Die Studienergebnisse zeigten, dass multiresistente E. cloacae Stämme genauso empfindlich gegenüber Desinfektionsmitteln waren wie der Typstamm. Organische Belastung interagierte stark mit Natriumhypochlorit und
minderte dadurch seine Wirksamkeit, während Peressigsäure und Ethanol nicht durch organische Verunreinigung beeinflusst wurden. Die Kontaktzeit hatte nur einen geringen Einfluss auf die bakterizide Wirkung. Im Gegensatz dazu spielten bei Benzalkoniumchlorid organische Verunreinigung und die Kontaktzeit eine wichtige Rolle. Insgesamt waren die minimalen Hemmkonzentrationen und die bakterizid wirksamen Konzentrationen niedriger als die für kommerzielle Produkte gebräuchlichen Konzentrationen. In den Keimträgertests hatte das Trocknen auf einer glatten Oberfläche einen Einfluss auf das Überleben eines Stammes von E. cloacae. Die Ergebnisse zeigten auch, dass sich die Wirksamkeit der Desinfektionsmittel in den verschiedenen verwendeten Tests deutlich unterscheiden kann. Die Ergebnisse waren schwer mit anderen Studien zu vergleichen, da eine internationale Durchführungsrichtlinie für die Prüfung der Wirksamkeit von Desinfektionsmitteln gegen multiresistente Bakterien fehlt.
Fazit: Peressigsäure, Ethanol, Benzalkoniumchlorid und Natriumhypochlorit eignen sich zur Desinfektion von multiresistenten E. cloacae. Die Wirksamkeit von Natriumhypochlorit und Benzalkoniumchlorid wird jedoch stark durch organische Stoffe beeinflusst. Dies unterstreicht die Bedeutung geeigneter Reinigungsmaßnahmen vor der Desinfektion. Wenn dies erfolgt ist, erweisen sich die getesteten Desinfektionsmittel gegen multiresistente E. cloacae genauso effektiv wie gegen den Typstamm.:1. Introduction ............................................................ 1
2. Literature Review ....................................................... 3
2.1 Enterobacteriaceae ..................................................... 4
2.1.1 General properties ................................................... 4
2.1.2 Enterobacter cloacae complex ......................................... 4
2.2 Multidrug-resistant bacteria and disinfectant “resistance” ............. 6
2.3 Disinfectant testing ................................................... 7
2.4 Active substances investigated in this study ........................... 8
2.4.1 Peracetic acid (PAA) ................................................. 8
2.4.2 Ethanol (ETH) ........................................................ 9
2.4.3 Benzalkonium chloride (BKC) .......................................... 9
2.4.4 Sodium hypochlorite (NaOCl) .......................................... 10
3. Materials and Methods ................................................... 11
3.1 Materials .............................................................. 11
3.2 Methods ................................................................ 14
3.2.1 Culture and storage of bacteria ...................................... 14
3.2.2 Preparation of disinfectants and the neutralizing agent .............. 14
3.2.3 Minimum inhibitory concentration (MIC) ............................... 15
3.2.4 Qualitative suspension test .......................................... 15
3.2.5 Quantitative suspension test.......................................... 16
3.2.6 Surface disinfection without mechanical action (germ carrier test) ... 16
3.2.7 Statistical analysis ................................................. 17
4. Results ................................................................. 18
4.1 Minimum inhibitory concentrations ...................................... 18
4.2 Qualitative suspension tests ........................................... 18
4.3 Quantitative suspension tests .......................................... 21
4.4 Surface disinfection without mechanical action (germ carrier test) ..... 24
5. Discussion .............................................................. 28
6. Summary ................................................................. 31
7. Zusammenfassung ......................................................... 33
8. References .............................................................. 35
9. Appendix ................................................................ 49
Acknowledgments ............................................................ 50 / Introduction: Enterobacter (E.) cloacae subsp. cloacae are important human pathogens, particularly in hospitalized patients. They tend to contaminate various medical devices and nosocomial outbreaks have been reported to be associated with the colonization of surgical equipment. Therefore, it is critical to determine the efficacy and effectiveness of disinfectants against this bacterial species.
Objectives: The current study was undertaken to prove whether single active ingredients (i.e. peracetic acid, ethanol, benzalkonium chloride, and sodium hypochlorite) of widely used commercial disinfectants provide proper efficacy against multidrug-resistant human isolates of E. cloacae.
Material and Methods: Six multidrug-resistant E. cloacae isolates obtained from patients in a clinical setting were tested and compared to the E. cloacae type strain. The studies were performed in vitro using peracetic acid, ethanol, benzalkonium chloride and sodium hypochlorite following the guidelines specified by the Disinfectants Commission within the Association of Applied Hygiene. Tests included determination of minimum inhibitory concentrations, bactericidal values by qualitative and quantitative suspension tests, and so-called germ carrier tests. The influence of exposure time and organic load on bacteriostatic and bactericidal concentrations was evaluated for each disinfectant using the two-tailed Mann-Whitney U-test.
Results: Study results showed that multidrug-resistant E. cloacae strains were equally susceptible to disinfectants as the type strain. Organic matter highly interfered with sodium hypochlorite thereby decreasing its efficacy whereas peracetic acid and ethanol were not influenced by organic soiling. Contact time had only a minor effect on bactericidal values. This was in contrast to benzalkonium chloride where organic soiling and contact time played an important role. On the whole, minimum inhibitory concentrations and bactericidal concentrations were lower than in-use concentrations of commercial products. Drying on smooth surfaces in the carrier tests had an effect on the survival of one E. cloacae strain. Results also showed that efficacious values determined by the different tests used may differ distinctly. Results were difficult to compare with other studies because an international practical standard for testing disinfectant efficacy against multidrug-resistant bacteria is missing.
Conclusion: Peracetic acid, ethanol, benzalkonium chloride and sodium hypochlorite are suitable to disinfect multidrug-resistant E. cloacae but the effectiveness of sodium hypochlorite and benzalkonium chloride is strongly influenced by organic matter. This underlines the importance of proper cleaning measures before disinfection. When this is done, the tested disinfectants proved to be as efficient against multidrug-resistant E. cloacae as against the type strain.:1. Introduction ............................................................ 1
2. Literature Review ....................................................... 3
2.1 Enterobacteriaceae ..................................................... 4
2.1.1 General properties ................................................... 4
2.1.2 Enterobacter cloacae complex ......................................... 4
2.2 Multidrug-resistant bacteria and disinfectant “resistance” ............. 6
2.3 Disinfectant testing ................................................... 7
2.4 Active substances investigated in this study ........................... 8
2.4.1 Peracetic acid (PAA) ................................................. 8
2.4.2 Ethanol (ETH) ........................................................ 9
2.4.3 Benzalkonium chloride (BKC) .......................................... 9
2.4.4 Sodium hypochlorite (NaOCl) .......................................... 10
3. Materials and Methods ................................................... 11
3.1 Materials .............................................................. 11
3.2 Methods ................................................................ 14
3.2.1 Culture and storage of bacteria ...................................... 14
3.2.2 Preparation of disinfectants and the neutralizing agent .............. 14
3.2.3 Minimum inhibitory concentration (MIC) ............................... 15
3.2.4 Qualitative suspension test .......................................... 15
3.2.5 Quantitative suspension test.......................................... 16
3.2.6 Surface disinfection without mechanical action (germ carrier test) ... 16
3.2.7 Statistical analysis ................................................. 17
4. Results ................................................................. 18
4.1 Minimum inhibitory concentrations ...................................... 18
4.2 Qualitative suspension tests ........................................... 18
4.3 Quantitative suspension tests .......................................... 21
4.4 Surface disinfection without mechanical action (germ carrier test) ..... 24
5. Discussion .............................................................. 28
6. Summary ................................................................. 31
7. Zusammenfassung ......................................................... 33
8. References .............................................................. 35
9. Appendix ................................................................ 49
Acknowledgments ............................................................ 50
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Comparison of recovery and enumeration of Escherichia coli, Cronobacter species, coliforms, and salmonella typhimurium in ground beef and ground turkey using conventional methods and a new chromogenic medium, ECA Check® Easygel® plusWenke, Erin Janet January 1900 (has links)
Master of Science / Food Science Institute / Daniel Y.C. Fung / ECA Check® Easygel® Plus (ECA) is a pectin-based gelling system that reacts with calcium ions bound to a pre-treated Petri dish, eliminating autoclaving prior to use. It can chromogenically and/or fluorogenically distinguish three organisms: Escherichia coli, Salmonella spp., and coliforms. This study compared the recovery of these organisms to conventional media using stock culture, inoculated, and non-inoculated ground beef and ground turkey. ECA was compared to Violet Red Bile Agar (VRB), Violet Red Bile Agar with 4-methylumbelliferyl-β-D-glucuronide (VRB-MUG), Xylose Lysine Desoxycholate Agar (XLD), Escherichia coli/Coliform (ECC) Count Plate Petrifilm™, and Tryptic Soy Agar (TSA). The stock culture recovery of Salmonella Typhimurium for ECA, TSA, and XLD were 8.62, 8.69, and 6.82 log CFU/ml, respectively. There was very little difference between the media in the recovery of Escherichia coli and Cronobacter spp., formerly referred to as Enterobacter sakazakii. Mean counts of presumptive E. coli in ground beef were 7.24 and 7.41 logs for ECA and VRB-MUG. Total coliform mean counts were 7.43, 7.63, and 7.37 logs for ECA, Petrifilm™, and VRB. Presumptive Salmonella means were 6.68 and 6.21 logs on ECA and XLD, while total aerobic counts were 7.84 and 6.51 logs on ECA and TSA. At 6.72 logs, ECA recovered considerably more Salmonella than XLD (5.71 logs) from the inoculated ground turkey; ECA recovered 7.62 logs total aerobic count which was significantly more than TSA at 6.89 logs. Total counts for both non-inoculated ground meats resulted in significant differences between TSA recovery and all other media. ECA also recovered significantly more than Petrifilm™ from both non-inoculated foods. The randomly selected organisms recovered from ECA were identified using BBL™ Crystal™ Enteric/Nonfermenter ID or Gram-Positive kits,
and correlated precisely to the chromogenic reaction of the colonies. ECA Check® Easygel® was efficient, less labor-intensive, comparable to, and, in some instances, better than conventional media at recovering target organisms.
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Detection of Enterobacter sakazakii in South African food productsKemp, Francisca 12 1900 (has links)
Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2005. / It is estimated by the World Health Organisation (WHO) that thousands of millions of
cases of foodborne diseases occur world–wide every year. Enterobacter sakazakii is
a member of the family Enterobacteriaceae and has been identified as an occasional
contaminant of powdered infant formula milk (IFM). Enterobacter sakazakii is an
opportunistic emerging pathogen and has the ability to cause a severe form of
neonatal meningitis. This organism was referred to as “yellow pigmented
Enterobacter cloacae” until 1980 after which it was renamed as E. sakazakii.
The current method for the detection of E. sakazakii is very time consuming
and includes pre–enrichment, enrichment in Enterobacteriaceae enrichment broth,
subsequent plating on violet red bile glucose agar and subculturing on tryptone soy
agar. In this study a polymerase chain reaction (PCR) method was developed for the
identification of the presence of E. sakazakii in infant food products. A part of the 16S
ribosomal RNA (rRNA) gene from E. sakazakii was amplified using the primer pair
Esak2 and Esak3.
An internal amplification control (IAC) was constructed as part of the PCR
detection method. The 850 base pair (bp) E. sakazakii PCR product was digested
with AluI and the two fragments containing the primer binding sites were ligated,
resulting in a 240 bp IAC. During this study a positive band for both the target DNA
(850 bp) and the IAC (240 bp) was simultaneously observed when the IAC was
added to the PCR mixture at a concentration of 0.72 pg.ml-1.
Four of 22 South African food products tested positive for the presence of
E. sakazakii, using both the PCR and recommended culturing methods. The PCR
method was used successfully for the detection of E. sakazakii within three days and
thus provides a possible alternative and improvement on the recommended current
culturing methods. Other microorganisms present in the products tested included
Escherichia coli, Klebsiella pneumoniae, Raoultella terrigena (“Klebsiella terrigena”)
and Chryseomonas luteola.
Since E. sakazakii is usually present in low numbers in food products, it is
possible that these few cells are unevenly distributed in the products, making it important to take multiple samples when evaluating IFM and thereby ensuring that
even low numbers of this pathogen are detected.
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A study of the chemical components of extracts from kirkia wilmsii and an investigation into their propertiesChigayo, K. 24 February 2015 (has links)
MSc (Chemistry) / Department of Chemistry
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Molecular perception and metabolic rewiring of the host plant by beneficial microbe Enterobacter sp. SA187Alzayed, Waad S. 10 1900 (has links)
Among abiotic stresses, salinity is considered the main limiting stress that negatively affects plant growth and reduces productivity worldwide. To overcome this challenge, a sustainable solution such as plant growth-promoting bacteria (PGPB) can be used to meet the increasing demand for food. The desert microbe Enterobacter sp SA187, an endophytic PGPB, induces salt tolerance in both model plant and crops. The interaction between SA187 and the host plant triggers the sulfur pathway in the bacteria which then provides multiple sulfur-containing compounds to its host plant. However, the molecular sensor of these compounds in the host plant is not known. Here, we show that SA187 activates the plant target of rapamycin (TOR) pathway. The beneficial effect of SA187 was lost in TOR mutants like raptor, and by the application of TOR inhibitor AZD8055. Next, we show that SA187 modulates the one- carbon (1C) metabolism of the host plant consisting of methionine and folate cycles. The beneficial effect of SA187 was compromised by using chemical inhibitors of folate cycle like Methotrexate (MTX) and Sulfadiazine (SDZ). The intermediates of the 1C metabolism like Homocysteine and S-adenosyl methionine (SAM) showed similar beneficial effects as SA187 colonized plants. Finally, we showed that SA187 enhances 1C metabolism activity by increasing methylation index (SAM/SAH ratio) in the plants. Taken together, we could show that host TOR-1C axis is essential for plant salt
tolerance by SA187.
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Detection, identification and live/dead differentiation of the emerging pathogen Enterobacter sakazakii from infant formula milk and the processing environmentCawthorn, Donna-Maree 12 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The World Health Organisation (WHO) estimates that at least 75% of infants receive
infant formula milk (IFM) either entirely or in conjunction with breast milk during the first
four months after birth. The presence of the emerging pathogen Enterobacter sakazakii
in IFM has been associated with rare but fatal cases of neonatal infections and deaths.
There is thus a need for accurate methods for the rapid detection of E. sakazakii in
foods. At present, the methods used to detect and identify this micro-organism are
inadequate, controversial and contradictory. The aim of this study was to determine the
most suitable method for E. sakazakii detection after evaluation of the currently
available methods. A further aim was to optimise a polymerase chain reaction (PCR)
method for the detection of only viable E. sakazakii cells utilising the DNA-intercalating
dyes ethidium monoazide (EMA) and propidium monoazide (PMA).
The Food and Drug Administration (FDA) method for E. sakazakii detection was
utilised to select 50 isolates from IFM and 14 from the environment, regardless of
colony appearance. These isolates were identified by sequencing a 1.5 kilobase (kb)
fragment of the 16S ribosomal DNA (rDNA) and by using the National Centre for
Biotechnological Information (NCBI) database to confirm the closet known relatives.
Seven of the 50 (14%) IFM isolates and six of the 14 (43%) environmental isolates were
identified as E. sakazakii. The methods that were evaluated for accuracy in detecting
and identifying these E. sakazakii isolates included yellow pigment production on
tryptone soy agar (TSA), chromogenic Druggan-Forsythe-Iversen (DFI) and
Enterobacter sakazakii (ES) agars and PCR using six different species-specific primer
pairs described in the literature.
The suitability of the FDA method was lowered by the low sensitivity, specificity
and accuracy (87%, 71% and 74%, respectively) of using yellow pigment production for
E. sakazakii identification. DFI and ES agars were shown to be sensitive, specific and
accurate (100%, 98% and 98%, respectively) for the detection of E. sakazakii. The
specificity of the PCR amplifications was found to vary between 8% and 92%, with
Esakf and Esakr being the most accurate of the primer pairs evaluated.
The current FDA method for E. sakazakii detection requires revision in the light of
the availability of more sensitive, specific and accurate detection methods. Based on
the results obtained in this study, a new method is proposed for the detection of
E. sakazakii in food and environmental samples. This proposed method replaces the
culturing steps on violet red bile glucose agar (VRBGA) and TSA with culturing on chromogenic DFI or ES agar. For identification and confirmation of presumptive
E. sakazakii isolates, the oxidase test, yellow pigment production and API biochemical
profiling is replaced by DNA sequencing and/or species-specific PCR with the most
accurate primer pair (Esakf and Esakr). The amendments to the current FDA method
will reduce the time to detect E. sakazakii from approximately 7 days to 4 days and
should prove to be more sensitive, specific and accurate for E. sakazakii detection.
In this study, a novel PCR-based method was developed which was shown to be
capable of discriminating between viable and dead E. sakazakii cells. This was
achieved utilising the irreversible binding of bacterial DNA to photo-activated PMA or
EMA in order to prevent PCR amplification from the dead cells. At concentrations of 50
and 100 μg.ml-1, PMA completely inhibited PCR amplification from dead cells, while
causing no significant inhibition of the PCR amplification from viable cells. EMA was
equally effective in preventing PCR amplification from dead cells, however, it also
inhibited PCR amplification from viable cells. PMA-PCR in particular, will be useful for
assessing the efficacy of processing techniques, as well as for monitoring the
resistance, survival strategies and stress responses of E. sakazakii. This will be an
important step in the efforts to eliminate E. sakazakii from food and food production
environments. / AFRIKAANSE OPSOMMING: Die Wêreld Gesondheidsorganisasie (WGO) beraam dat ten minste 75% van alle babas
net baba formule melk (BFM) of BFM in kombinasie met moedersmelk in die eerste vier
maande na geboorte kry. Die teenwoordigheid van die voortkomende patogeen
Enterobacter sakazakii in BFM is al geassosieer met skaars maar noodlottige gevalle
van neonatale infeksies en sterftes. Akkurate metodes word dus benodig vir die vinnige
deteksie van E. sakazakii in voedsel. Die metodes wat huidiglik gebruik word vir die
deteksie en identifikasie van hierdie mikroörganisme is onvoldoende, kontroversieël en
teenstrydig. Die doel van hierdie studie was om die beste metode vir die deteksie van
E. sakazakii te bepaal, na 'n evaluasie van die metodes wat huidiglik beskikbaar is. 'n
Verdere doel was om 'n polimerase ketting reaksie (PKR) metode vir die deteksie van
slegs lewensvatbare E. sakazakii selle te optimiseer deur gebruik te maak van die DNSbindende
kleurstowwe, etidium mono-asied (EMA) en propidium mono-asied (PMA).
Die Voedsel en Medisyne Administrasie (VMA) se metode vir E. sakazakii deteksie
is gebruik om, ongeag van die kolonie kleur, 50 isolate vanuit BFM en 14 isolate vanuit
die omgewing te kies. Hierdie isolate is geïdentifiseer deur die DNS volgorde van 'n 1.5
kilo-basis (kb) fragment van die 16S ribosomale DNS (rDNS) te bepaal en die Nationale
Sentrum vir Biotegnologiese Informasie (NSBI) databasis te gebruik om die mees
verwante spesie te bevestig. Sewe van die 50 (14%) BFM isolate en ses van die 14
(43%) omgewings isolate is geïdentifiseer as E. sakazakii. Die metodes wat geëvalueer
is in terme van akkuraatheid vir deteksie en identifikasie van hierdie E. sakazakii isolate
het PKR met ses verskillende spesie-spesifieke peiler pare soos beskryf in die
literatuur, geel-pigment produksie op triptoon soja agar (TSA) en chromogeniese
Druggan-Forsythe-Iversen (DFI) en Enterobacter sakazakii (ES) agars ingesluit. Die
geskiktheid van die VMA metode is verlaag deur die lae sensitiwiteit, spesifisiteit en
akkuraatheid (87%, 71% en 74% onderskeidelik) van geel pigment produksie vir
E. sakazakii identifikasie. Chromogeniese DFI en ES agars was sensitief, spesifiek en
akkuraat (100%, 98% en 98% onderskeidelik) vir die identifikasie van E. sakazakii. Die
spesifisiteit van die PKR produkte het gewissel tussen 8% en 92%, en Esakf en Esakr is
as die akkuraatste geëvalueerde peiler paar geidentifiseer.
Die huidige VMA metode vir E. sakazakii deteksie vereis hersiening aangesien
meer sensitiewe, spesifieke en akkurate deteksiemetodes voortdurend beskikbaar
word. 'n Nuwe metode, gebaseer op die resultate van hierdie studie, word voorgestel
vir die deteksie van E. sakazakii in voedsel- en omgewingsmonsters. Die voorgestelde metode vervang die kwekingsstap op violet rooi gal glukose agar (VRGGA) en TSA
deur kweking op chromogeniese DFI of ES agars. Verder word die oksidase toets, geel
pigment produksie en API biochemiese profiele van vermoeidelike E. sakazakii isolate
vervang deur DNS volgorde bepaling en/of spesie-spesifieke PKR met die mees
spesifieke peiler paar (Esakf and Esakf) vir die identifikasie en bevestiging van
E. sakazakii. Die voorgestelde wysigings van die VMA metode sal die tydsduur van
E. sakazakii identifikasie van 7 dae na 4 dae verminder, en behoort ook meer sensitief,
spesifiek en akkuraat te wees vir die deteksie van E. sakazakii.
'n Nuwe PKR-gebaseerde metode wat tussen lewensvatbare en dooie
E. sakazakii selle kan onderskei is in hierdie studie ontwikkel. Dit is bereik deur die
onomkeerbare binding van bakteriële DNS aan lig-geaktiveerde EMA of PMA om die
PKR amplifisering van dooie selle te voorkom. Konsentrasies van 50 en 100 μg.ml-1
PMA het PKR amplifikasie heeltemal geïnhibeer, terwyl geen inhibisie van
lewensvatbare selle bespeur kon word nie. EMA was ook suksesvol in die voorkoming
van die PKR amplifikasie van dooie selle, alhoewel daar ook 'n mate van DNS inhibisie
was tydens die amplifikasie van lewensvatbare selle. PMA-PKR kan ook van nut wees
vir die assessering van die doeltreffendheid van prosesseringstegnieke, en ook vir die
waarneming van die weerstandigheid, oorlewingsstrategieë en stresresponse van
E. sakazakii. Dit sal 'n belangrike stap wees in pogings om E. sakazakii van voedsel en
voedsel produksieomgewings te elimineer.
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Phylogeny and molecular identification of Cronobacter strains isolated from south African food productsStrydom, Amy 03 1900 (has links)
Thesis (MSc Food Sc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The genus Cronobacter (Enterobacter sakazakii) contains opportunistic pathogens that can
cause a severe form of neonatal meningitis, necrotising enterocolitis and septicaemia.
Cronobacter infections have been reported in all age groups, however, immunocompromised
infants are more susceptible to these infections. Furthermore, Cronobacter
strains have been reported to show differences in sensitivity to antibiotics and virulence.
These differences led to the reclassification of Cronobacter and currently the genus
contains five distinct species, namely Cronobacter sakazakii, Cronobacter malonaticus,
Cronobacter turicensis, Cronobacter dublinensis and Cronobacter muytjensii. As this
reclassification was only accepted recently, there are not many typing methods optimised
for differentiation between the five Cronobacter species. Typing of Cronobacter strains are
important as the species may be diverse regarding their virulence.
Cronobacter strains have been isolated from infant formula milk (IFM), the
environment of an IFM processing facility and fresh produce in South Africa. However,
little is known about the phylogeny and prevalence of these strains. The aim of this study
was to classify 24 South African Cronobacter strains (previously identified as E. sakazakii)
and to evaluate the phylogeny of the isolates based on the 16S ribosomal RNA (rRNA) and
rpoA genes. All 24 South African strains were identified as Cr. sakazakii despite a wide
variety of isolation sources. Other studies have also found that irrespective of the isolation
source, the majority of Cronobacter strains are identified as Cr. sakazakii. The South
African strains were found to be phylogenetically closely related. However, two distinct
clusters separated at a 93 % confidence level were observed in the Cr. sakazakii group
based on the 16S rRNA gene analysis.
Strains of Cr. sakazakii, Cr. dublinensis, Cr. turicensis and Cr. muytjensii were
differentiated from each other with sequence data of the 16S rRNA and rpoA genes, but it
was not possible to differentiate between Cr. sakazakii and Cr. malonaticus. The
phylogram based on the rpoA gene sequences did separate Cr. malonaticus and Cr.
sakazakii strains, however, the clusters were separated with a low bootstrap value of 70 %.
Phylogenetic analysis based on the rpoA and 16S rRNA genes were, therefore, not
sufficient to distinguish between all the Cronobacter species. The sequence data of these
two genes can be used to differentiate between the Cronobacter strains when used in
combination with malonate utilisation analysis.
A PCR-RFLP method was subsequently developed to facilitate the simultaneous
differentiation between all five Cronobacter species. The PCR-RFLP assay was based on
the amplification of the rpoB gene followed by the combined digestion with restriction
endonucleases Csp6I and HinP1I. Unique profiles for each of the five Cronobacter species
were obtained and it was also possible to differentiate between Enterobacteriaceae and
Cronobacter strains. Furthermore, two strains which were identified as Cr. sakazakii with
sequencing based on the 16S rRNA and rpoA genes had PCR-RFLP profiles identical to
that of Cr. malonaticus. Sequencing based on the rpoB gene and additional biochemical
analysis with malonate broth confirmed the identities of these two strains as Cr.
malonaticus. This PCR-RFLP assay is, therefore, an accurate typing method that ensures
rapid differentiation between the five species of Cronobacter. / AFRIKAANSE OPSOMMING: Die Cronobacter genus (Enterobacter sakazakii) bevat opportunistiese patogene wat 'n
ernstige vorm van neonatale meningitis, enterokolitis en septisemie kan veroorsaak.
Cronobacter infeksies is al in alle ouderdomsgroepe aangemeld, maar
immuungekompromitteerde babas is die meeste vatbaar vir hierdie infeksies. Verder toon
Cronobacter spesies verskille in virulensie en sensitiwiteit vir antibiotika. Hierdie verskille
het gelei tot die herklassifikasie van Cronobacter en tans bestaan die genus uit vyf
afsonderlike spesies, naamlik Cronobacter sakazakii, Cronobacter malonaticus,
Cronobacter turicensis, Cronobacter dublinensis en Cronobacter muytjensii. Aangesien
hierdie herklassifikasie slegs onlangs aanvaar is, is daar nie baie metodes wat geskik is vir
onderskeiding tussen die vyf Cronobacter spesies nie. Onderskeiding tussen Cronobacter
spesies is belangrik omdat die spesies verskillend kan wees met betrekking tot hulle
virulensie.
Cronobacter is geisoleer uit baba formule melk (BFM), die omgewing van 'n BFM
fabriek en vars produkte in Suid-Afrika. Daar is egter nie baie bekend oor die filogenie en
voorkoms van hierdie isolate nie. Die doel van hierdie studie was om 24 Suid-Afrikaanse
Cronobacter stamme (voorheen geïdentifiseer as E. sakazakii) te klassifiseer en die
filogenie van die isolate te evalueer gebaseer op die 16S ribosomale RNS (rRNS) en rpoA
gene. Al 24 Suid-Afrikaanse stamme is geïdentifiseer as Cr. sakazakii ten spyte van 'n
wye verskeidenheid isolasie bronne. Ander studies het ook gevind dat, ongeag die isolasie
bron, die meerderheid van Cronobacter stamme as Cr. sakazakii geïdentifiseer word. In
hierdie studie is gevind dat die Suid-Afrikaanse stamme filogeneties nou verwant is. Op
grond van die 16S rRNA geen analise is die Cr. sakazakii stamme egter in twee
afsonderlike groepe gedeel met 'n 93% vertrouens vlak.
Dit was moontlik om stamme van Cr. sakazakii, Cr. dublinensis, Cr. turicensis en Cr.
muytjensii van mekaar te onderskei met die DNS volgorde data van die 16S rRNA en rpoA
gene, maar geen onderskeid tussen Cr. sakazakii en Cr. malonaticus stamme was
moontlik nie. Die filogram gebaseer op die rpoA DNS volgorde data het aparte takke vir Cr.
malonaticus en Cr. sakazakii stamme getoon, maar die twee takke is met ‘n lae vertrouens
waarde van slegs 70 % geskei. Filogenetiese analise gebaseer op die rpoA en 16S rRNA
gene is dus nie voldoende om te onderskei tussen al die Cronobacter spesies nie. Die
DNS volgorde data van hierdie twee gene sou egter gebruik kon word om te onderskei
tussen die Cronobacter spesies wanneer dit gebruik word in kombinasie met
malonaatbenutting-analises.
'n Polimerase ketting reaksie (PKR) beperkings fragment lengte polimorfisme
(BFLP) metode is ontwikkel om die gelyktydige onderskeiding tussen al vyf Cronobacter
spesies te fasiliteer. Die PKR-BFLP metode is gebaseer op die vermeerdering van die
rpoB geen gevolg deur die gesamentlike vertering met die beperkingsensieme, Csp6I en
HinP1I. Unieke profiele vir elk van die vyf Cronobacter spesies is verkry en dit was ook
moontlik om tussen Enterobacteriaceae en Cronobacter spesies te onderskei. Verder het
twee stamme wat as Cr. sakazakii geïdentifiseer is met DNS volgordebepaling van die 16S
rRNA en rpoA gene, PKR-BFLP profiele identies aan dié van Cr. malonaticus getoon.
DNS volgordebepaling van die rpoB geen en ‘n addisionele biochemiese toets met
malonaat sop het die identiteit van hierdie twee stamme as Cr. malonaticus bevestig.
Hierdie PKR-BFLP is dus 'n akkurate metode wat vinnige onderskeid tussen die vyf
spesies van Cronobacter kan verseker.
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Impact de la ventilation mécanique sur la réponse inflammatoire médiée par les Toll-like receptors 2 et 4 dans un modèle de pneumopathie bactérienne / Impact of mechanical ventilation on inflammatory response mediated by Toll-like Receptors 2 and 4 in a model of bacterial pneumoniaBarbar, Saber Davide 28 October 2014 (has links)
Introduction: La pneumonie associée à la ventilation mécanique (VM) est fréquente chez les patients ventilés. L’étirement cyclique (EC) induit par la VM pourrait amorcer le poumon vers une réponse inflammatoire en cas d'exposition à des bactéries. Les Toll-like Receptors (TLR) reconnaissent les bactéries et déclenchent l'immunité. La VM pourrait moduler l'expression des TLR et leur réactivité aux agonistes. Le décubitus ventral (DV) réduit l’étirement du poumon. Méthodes: Les niveaux de TLR2 et la réponse à ses agonistes ont été mesures dans des cellules pulmonaires soumises à un EC, et dans un modèle de lapin ventilé. Une stimulation ex vivo du sang total prélevé sur lapins ventilés a été réalisée. Une pneumonie a été induite chez des lapins soumis à VM et maintenus en décubitus dorsal ou tournés en DV. Résultats: L’EC des cellules ainsi que des poumons de lapins augmente les niveaux de TLR2 et la réponse inflammatoire à ses agonistes. La VM et l’exposition du poumon à des agonistes TLR2 induisent synergiquement des lésions. Chez des lapins avec pneumonie sous VM la clairance bactérienne pulmonaire est réduite, la probabilité de bactériémie et le taux des cytokines circulantes augmentés. Le sang total provenant d'animaux sous VM libère de grandes quantités de cytokines après stimulation. Le DV est associe à des niveaux plus faibles de concentrations bactériennes et d'inflammation. Conclusions: La VM sensibilise le poumon aux ligands bactériens de TLR2, modifie la clairance bactérienne pulmonaire, favorise les lésions pulmonaires et de l'inflammation. La surexpression de TLR2 induite par l’EC pourrait expliquer ces différences. Le DV pourrait avoir un effet protecteur. / Introduction: Ventilator-associated pneumonia is common in patients subjected to mechanical ventilation (MV). Cyclic stretch subsequent to MV could prime the lung toward an inflammatory response if exposed to bacteria. Toll-like receptors (TLRs) recognize pathogens thus triggering immunity. MV could modulate TLRs expression and responsiveness to agonists. The prone position (PP) reduces lung stretch.Methods:TLR2 levels and response to the TLR2 ligands were measured in human pulmonary cells submitted to cyclic stretch, and either spontaneously breathing (SB) or MV rabbits. Ex vivo stimulation of whole blood taken from SB or MV rabbits was performed.Enterobacter aerogenes pneumonia was induced in rabbits subjected to MV and kept supine or turned to the PP. Results: Cyclic stretch of human cells as well as rabbitsÕ lung increased both TLR2 levels and inflammatory response to its agonist. MV and airways exposure to TLR2 ligands acted synergistically in causing lung injury.A decrease of lung bacterial clearance and a greater likelihood of bacteremia were observed in MV rabbits with S. aureus pneumonia. Circulating cytokines rose significantly only in these animals. MV induced TLR2 spleen overexpression. Whole blood obtained from MV animals released larger amounts of cytokines after stimulation. PP was associated with lower levels of bacterial concentrations and inflammation. Conclusions: MV sensitizes the lung to bacterial TLR2 ligands, alters lung bacterial clearance, promotes lung injury and inflammation. Both pulmonary and peripheral blood stretch-induced TLR2 overexpression could account at least in part for such differences. The PP could be protective.
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Biochemical Characterization of β-galactosidase from Enterobacter sp. YSUMotari, Fred Ondabu 02 May 2023 (has links)
No description available.
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Quantitative determination of selenite transformation by <i>Enterobacter</i> sp. YSU and <i>Stenotrophomonas maltophilia</i> OR02Shaik, Nabeel A. January 2011 (has links)
No description available.
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