Características Socio Económicas de la Población

Cronobacter malonaticus is a member of the genus Cronobacter which is considered an

opportunistic pathogen. C. malonaticus has been recognised to be more associated with adult infections, whereas the closely related species C. sakazakii has been reported to be responsible for several serious neonatal infections. Therefore, research has been focused on C. sakazakii to investigate its physiological and virulence traits. However, the significance of C. malonaticus has recently increased since it was documented to be involved in several severe neonatal infections. Unlike previous Cronobacter studies, this study is mainly focused on C. malonaticus to investigate its physiological and virulence characteristics that enable this species to survive different stresses and cause adult and neonatal infections.

 In the first part of this study a unique clinical collection of Cronobacter strains were used. The collection has been isolated from about eight different departments in two hospitals of the Czech Republic and isolates were collected from different clinical sites over a 6-year period. As the majority of the collected strains were isolated from adults, it was hypothesised that most of these strains could possibly be C. malonaticus. Also some strains were collected from the same department, thus it is worthy to study if there is any relatedness between strains. Therefore, a number of phenotypic and genotypic identification techniques have been applied to answer some questions such as 1) is C.

malonaticus the predominant species among this collection, 2) is there any link between

age group and a specific species, 3) is there any relatedness between strains, and finally 4) the study will find the effectiveness of each technique.

The use of some media such as TSA, VRBGA and DFI in laboratories does not give a complete reliable identification; however, they are still essential for growing and isolation of different bacteria and also for minimising the possible identification of microorganism in this level. Other phenotypic methods which have been applied in this study such as API ID32E showed also some limitations that confuse the user to reach a correct identification and could not speciate the strains. Therefore, genotypic methods such as rpoB, MLST, O-antigen and PFGE were applied. The 51 strains were predominated by C. sakazakii ST4 (63 %, 32/51) and C.

malonaticus ST7 (33 %, 17/51). These had been isolated from throat and sputum samples

complete agreement between O-antigen typing and rpoB gene sequence analysis and MLST profiling. The majority (42/51) of strains were from the respiratory system (i.e. throat swabs and sputum samples) and only three were from faeces, two from wound, one from blood and one from urine. Despite the high clonality of Cronobacter, PFGE profiles differentiated strains within each sequence type into 15 pulsotypes. This study shows the value of applying MLST to bacterial population studies with strains from two patient cohorts, combined with PFGE for further discrimination of strains.

 The second part of the study focused on comparative physiological and virulence related tests of 20 strains of different sequence types of C. malonaticus. Six ST7 strains were selected to represent the 5 C. malonaticus pulse types of the first part of this study and an additional 14 C. malonaticus strains were chosen from the Cronobacter PubMLST database from different countries to represent different STs.

As shown in figure 4.1 and 4.2 temperature was a crucial factor in biofilm formation, in addition to nutrient type present in milk is important in enhancement of biofilm formation. Capsules, cellulose and curli fimbriae were not confirmed to have a role in biofilm formation. Just four C. malonaticus strains, which are 1830, 1833, 1835 and 2020 were not able to show any movement activity. These strains share same DNA nucleotides and amino acid sequences for flhC gene.

In general, though there was a variation in capsule production between used strains, production of capsule was enhanced when IF was used. These findings may confirm the effect of ingredients of different medium on production of bacterial exopolysaccharides. The production of such capsular materials could be important in desiccation persistence, serum resistance and macrophage evasion (Ogrodzki and Forsythe, 2015). All C.

malonaticus showed an ability of producing cellulose in different amounts. Although C. malonaticus possesses curli fimbriae associated genes not all C. malonaticus strains were

able to express the curli fimbriae.

The majority of C. malonaticus strains showed the ability to survive low pH (3.5 pH) over 2 hours of incubation. Just ST60 strains declined by more than 2 logs at the last time point of incubation. While just one strain, 685, which belongs to ST129 showed no ability to survive directly after 30 minutes. Although the ompR and rpoS genes were detected in all C.

malonaticus, strain 685 showed to have different nucleotide sequence of these two genes.

This difference could effect the function of these genes and thus this strain become less tolerant to the acidic environment.

The recovery of sublethally injured C. malonaticus strains was decreased comparing with the number of inoculum bacteria. However, the number of non-detected bacteria on VRBGA was higher than on TSA. This reflects the differences in composition between the two culture media. Interestingly, the lowest recovery of injured cells was showed by strain 685. Exceptionally, this strain was not able to survive acidic stress after 30 minutes. This strain was shown to have different nucleotides sequence of two genes, ompR and rpoS, that associated with stress response. In general, C. malonaticus has been frequently isolated from PIF and other dry and desiccated environments (Cronobacter MLST Pub Databases) which suggest that C. malonaticus can survive the dry conditions. Such a trait could make this microorganism a potential pathogen from dry sources such as PIF.

Some metals such as copper, zinc, iron, nickel and cobalt, manganese are essential in the structure of bacterial proteins; nonetheless these heavy metals could be toxic for bacteria even at low concentrations (Osman and Cavet, 2011). All tested strains showed an ability to tolerate or adapt the majority of the used metals at different concentrations. Nevertheless, C. malonaticus strains were sensitive to sodium tellurite at all used concentrations. This susceptibility was explained when all strains were confirmed in this study to lack the genes associated with tellurite resistance, terACDYZ. Pathogenic bacteria have an ability to control heavy metal homeostasis which help them to survive the killing action of macrophages. The control of heavy metals such as copper and zinc are involved in the ability of pathogenic microorganisms to survive into macrophages (Reva and Bezuidt, 2012). Noticeably, not all strains that showed ability to resist or adapt heavy metals were able to survive macrophages. This is more likely due to contribution of different factors in such activity. The iron acquisition system encoded in the genome of the sequenced of the

C. malonaticus strains, along with the ability of these strains to produce iron siderophores in vitro, are important to maintain its growth in vitro. This might also enhance its growth in vivo, and enable C. malonaticus to acquire iron in iron-limited environments in the human

Gram-negative bacteria which cause systemic infections need to overcome the mechanisms of killing by serum components and the action of the human complement in the human serum. In this study, despite the fact that some C. malonaticus strains were sensitive to human serum as well as rapidly killed by phagocytes, some other strains such as 688, 1827, 2018 (ST7) and 1569 (ST307) were serum tolerant and were able to survive and multiply within macrophages in the laboratory assays. This allows C. malonaticus to enhance their ability to grow in the bloodstream and thus this could be an advantage of causing bacteremia. Moreover, C. malonaticus was able to produce haemolysins and protease. The presence of such virulence factors indicates the possible occurrence of serious destruction in the human tissue during infections.

Though C. malonaticus strains harbour multiple drug resistance operon, mar, C.

malonaticus strains showed susceptibility to quinolones, aminoglycosides and

carbapenems groups. In contrast, all strains were resistant to tetracycline and 40 % of them were resistant to chloramphenicol. About 35% of C. malonaticus strains showed intermediate resistance to cefotaxime which is one of the third-generation of cephalosporins, related to penicillin.

Plasmid profiling was performed, using simpe experiment, on 20 C. malonaticus strains including two C. sakazakii strains BAA-894 and 6, which used as a reference marker and a negative control respectively. All of the strains contain at least one plasmid. However, because of the limitations of the experiment, it was impossible to determine the numbers and sizes of the plasmids.

 The third part of this study concentrated on the pathogenicity of the same strains that were used in the second part. The results in Figures 5.4, 5.7, 5.18 and Table 5.2 confirm that C.

malonaticus is potentially capable of causing serious infections such as NEC, bacteraemia

and meningitis. Two ST7 strains which are 1827 and 2018 were always high in their ability to invade and survive the Caco-2, HBMEC and macrophage cells (Table 5.2). Moreover, these two strains were resistant to human serum as shown in Chapter 4, resulting in bacteremia. These strains were isolated from blood and sputum respectively and were clustered together when PFGE was performed in Chapter 3. The CDC strain 1569 (ST307) which was isolated from the blood of a fatal neonatal case showed also significant results when it was able to invade Caco-2 at high level, invade HBMEC at moderate level and

survive the human macrophages over the 72 hours of incubation and replicate during the 48 hours (Table 5.2). This strain showed also moderate levels of serum resistance in Chapter 3. The result of the CDC strain 1569 confirm its responsibility for the fatal neonatal case in the USA.

Table 5.2 showed a summary of the ability of the C. malonaticus strains to adhere and invade the A549 respiratory and the T24 urinary cells. The two ST7 strains 1827 and 2018 which showed significant results when tested against Caco-2, HBMEC and macrophages showed also notable invasion results when they tested against A549 and T24. In addition, strain 1558 (ST7) showed also remarkable invasion levels into A549 and T24 cells (Table 5.2). However, majority of the tested strains showed higher ability to invade respiratory and urinary cells than other used cells. This might explain why C. malonaticus more associated with adult infections than neonatal infections. It's noteworthy that four ST7 strains (1830, 1833, 1835 and 2020), which were not motile and not able to produce any capsular materials, were always not able to invade the used cell lines or survive the macrophages, although they were isolated from throat and faeces. This indicates that C.

malonaticus could colonise the human intestine and throat without having abilities to cause

infections, presuming that they inhabit the human body as normal flora.

Finally, the results of this study proved the ability of C. malonaticus to overcome several stresses that could be faced either in the general environments such dry and acids or in host body such as acids, action of serum and toxic effects of metals. In addition, C.

malonaticus showed a great ability to invade and survive different human cell lines. These

findings, taken together, confirm the potential of C. malonaticus to cause serious infections in neonates or adults.