# New Parameters to Quantitatively Express the Invasiveness of Bacterial Strains from Implant-Related Orthopaedic Infections into Osteoblast Cells

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## Abstract

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## 1. Introduction

## 2. Materials and Methods

#### 2.1. Bacterial Strains

#### 2.2. Preparation of the Bacterial Inoculum for Cell Invasion Assays

#### 2.3. MG63 Cell Culture

#### 2.4. Invasion Assay of Osteoblasts in 96-Well Plates

^{3}cells/well, in a volume of 100 μL of MEM growth medium without antibiotics. The seeded plates were incubated at 37 °C under standard cell culture conditions. After 24 h of culture, MG63 cells were found to closely approximate a density of 1 × 10

^{4}cells/well [4]. Prior to exposing the osteoblast-like cells to the bacterial inoculum the wells of each plate were washed once with 200 μL of Dulbecco’s phosphate buffered saline (D-PBS, Sigma-Aldrich). One hundred microlitres of the initial bacterial suspension and its dilutions prepared as described above were added to triplicate wells, following the scheme illustrated in Figure 1.

^{2}:1 to 0.001:1 MOI (5 logs) for S. aureus and 10

^{4}:1 to 10:1 for less internalising species such as S. lugdunensis. Only MOI titres that gave a rate of internalization higher than 0 CFU were finally considered in the analyses.

#### 2.5. Statistics

_{10}MOI and Log

_{10}CFU vs. Log

_{10}MOI were achieved using the software Excel (Microsoft). Statistical comparison of the datasets by parametric (ANOVA followed by Bonferroni/Dunn test) and non-parametric (Kruskal–Wallis test followed by Kruskal–Wallis rank) tests was performed by StatView (version 5.0.1, Sas Institute Inc., Cary, NC, USA).

## 3. Results

#### 3.1. PIB Values vs. Inoculum Size (MOI)

#### 3.2. Internalised CFU vs. MOI Regression Curves

#### 3.3. New Parameters for Describing the Internalization Efficiency

_{10}(internalised CFU) = A × Log

_{10}(MOI) + B

^{4}eukaryotic cells internalise a single bacterium, i.e., the lowest concentration at which internalization occurs under the test conditions used. It has to be pointed out that this type of investigation requires the testing of numerous MOI titres in triplicate. This is feasible with our technique based on a 96-well microplate using 10

^{4}eukaryotic cells per well, but it would be rather laborious with culture plates with a reduced number of wells (up to nine six-well plates would be required for testing a single strain).

_{10}(internalised CFU) = 0. Solving the equation, the IMI value is obtained as follows:

_{10}(internalised CFU) = 0 = A × Log

_{10}(MOI) + B

_{10}(MOI) = −B/A = Log

_{10}IMI = LIMI

^{4}osteoblasts), Log

_{10}(MOI) = 0. Once substituting the value into the equation:

_{10}(internalised CFU) = A × 0 + B = B

_{10}(internalised CFU) = B = LI1M

#### 3.4. Invasiveness of S. aureus Strains

^{4}CFU for LI strains; (iii) I1M measures suggest that, with an inoculum of 10

^{4}CFU (1:1 MOI) over 890 bacteria are taken up by osteoblasts in the case of HI strains against not even 1 for LI strains; (iv) LIMI and LI1M are parameters of less immediate interpretation, but both show a markedly decreased coefficient of variation with respect to PIB, IMI and I1M, thus resulting more robust parameters to be used as measures of invasiveness; and (v) LIMI is a logarithmic measure inversely proportional to the invasiveness of the strain and ranged from about −3 for the HI strains (with a C.V. of just 12% vs. 74% for PIB, 83% for IMI and 74% for I1M) to 0.6 for the LI strains (C.V. of 33% vs. 44% for PIB, 46% for IMI and 44% for I1M). LI1M assumes a value of 0 when with a 1:1 MOI inoculum a single bacterium is taken up by osteoblasts and of 4 when the entire inoculum is taken up. LI1M was of about 2.8 for HI strains and of −0.6 for LI strains (C.V. was similar to that found for LIMI). When the results concerning LIMI and LI1M were statistically analysed, the Bonferroni/Dunn test gave a p-value less than 0.0001 when comparing HI and LI strains.

#### 3.5. Invasiveness of S. epidermidis Strains

#### 3.6. Invasiveness of S. lugdunensis Strains

#### 3.7. Invasiveness of E. faecalis Strains

## 4. Discussion

^{4}osteoblasts are needed for a single event of internalization to occur. This finding suggests an impressive lack of interaction of this bacterial species with osteoblasts.

## 5. Conclusions

## Acknowledgments

## Author Contributions

## Conflicts of Interest

## References

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**Figure 1.**Scheme showing the organisation of the multiwell plates seeded with MG63 cells and subsequently treated with serial dilutions of bacterial suspensions. In the case of species incompetent for internalization, 10 dilutions of the starting bacterial suspensions were sufficient to reach levels with total absence of internalised bacteria and, thus, two bacterial strains could be contemporarily run on the same plate. Conversely, with S. aureus, greater dilutions needed to be reached.

**Figure 2.**Gentamicin protection assay protocol utilised to assess bacterial internalization in MG63 cells.

**Figure 3.**Variation of PIB (per cent of internalised bacteria) values obtained at different MOI (multiplicity of infection): (

**a**) plot showing the distribution of each PIB value as a function of the inoculum size for all six S. aureus isolates; and (

**b**) corresponding plot concerning three different S. epidermidis strains (those tested in three independent experiments). The R-square values of the PIB vs. MOI regression curve achieved for each single strain are reported in both graphs. It may be noticed as R-square values are generally very low and, in the broad range of MOI examined, any effect associated to the MOI appears negligible, especially in view of the large uncertainty of PIB measurements.

**Figure 4.**Correlation of CFU vs. MOI on semi-logarithmic scale. The regression curves of internalised CFU as a function of the 10-base logarithm of MOI are shown for all S. aureus strains.

**Figure 5.**Correlation of internalised CFU vs. MOI. This dispersion plot based on a logarithmic scale shows the first-order linear regression curves obtained for each of the nine S. aureus strains. Per each strain, the number of experiments performed, the equation of the regression curve and the R-square value are reported. For S. aureus strain cra1199, only the three experiments performed on the low scale of MOI are plotted. When including the additional two experiments performed for cra1199 on the top of the scale of MOI, the R-square was found to increase from the value reported here of 0.85 to 0.97.

**Figure 6.**Bacterial invasiveness expressed in terms of LI1M: (

**a**) LI1M value calculated for each S. aureus strain (HI S. aureus strains are in red, while LI strains are in light blue); and (

**b**) LI1M value calculated for each of the seven S. epidermids strains.

**Figure 7.**Different invasiveness across all investigated bacterial species expressed in terms of: (

**a**) PIB; (

**b**) I1M; and (

**c**) IMI. Bars represent mean values ± S.D. Bar graphs with PIB and I1M values consistently show the gap between HI S. aureus strains and the LI strains and the strains of all other species. In the case of IMI, the higher is the internalization minimal inoculum. The lower is the invasiveness. Thus, IMI could also be interpreted as a measure of the resistance to phagocytosis. Here, even when compared to other incompetent species, S. lugdunensis exhibits the highest IMI and, therefore, emerges as the species with the lowest uptake by osteoblasts.

**Figure 8.**LI1M (

**a**); and LIMI (

**b**) values across the different microbial species investigated. Bars represent mean values ± S.D.

**Figure 9.**Log CFU vs. Log MOI linear regression curves: S. epidermidis strains (

**a**); and S. lugdunensis strains (

**b**).

**Table 1.**Current parameters in use for expressing the potential of invasiveness of bacterial strains.

Acronym | Description | Meaning |
---|---|---|

NIB | Number of internalised bacteria | Number of internalised bacteria (at a given MOI). Usually expressed in terms of CFU per well or CFU per number of eukaryotic cells, NIB is easily obtained, but its use presents limitations. Its value is influenced by the MOI in use. The assessment of internalization of bacteria belonging to different species is usually performed at different MOI, thus direct comparison of NIB among strains is hampered. Obstacles are met when strains of the same species exhibit remarkable differences in invasiveness, ideally needing different inoculum MOI. |

PIB | Per cent of internalised bacteria | PIB value represents the per cent fraction of the inoculum that is taken up by the eukaryotic cells. Its value is directly proportional to the invasiveness of the test strain towards a specific eukaryotic cell type. Based on our current results, PIB is not significantly affected by MOI and is more appropriate than NIB for strains comparison. |

Species | Strain | Ribogroup | MLST CC | spa Type | spa CC | Origin |
---|---|---|---|---|---|---|

S. aureus | ATCC 25923 | - | CC30 * | t021 | CC021/012 | Clinical |

cra1733 | cra-119-S-8 | K | ||||

cra2727 | H | |||||

cra1772 | t298 | IF | ||||

cra1199 | t012 | EF | ||||

cra1451 | cra-138-S-2 | H | ||||

cra1611 | IF | |||||

cra1607 | H | |||||

cra1212 | cra-53-S-7 | EF |

Species | Strain | Ribogroup | Origin |
---|---|---|---|

S. epidermidis | cra1379 | cra-63-S-7 | FI |

cra1231 | H | ||

cra1275 | cra-63-S-4 | PSI | |

cra1141 | cra-92-S-5 | H | |

cra1145 | cra-122-S-2 | H | |

cra1378 | cra-119-S-4 | H | |

cra1428 | cra-80-S-1 | K | |

S. lugdunensis | cra1871 | cra-62-S-1 | FI |

cra1363 | FI | ||

cra2363 | cra-64-S-8 | FI | |

cra2501/1 | FE | ||

cra1750 | cra-74-S-5 | No MD | |

cra1871 | cra-62-S-1 | FI | |

E. faecalis | cra2174 | cra-116-S-1 | H |

cra1705 | cra-115-S-8 | FI |

Acronym | Description | Meaning |
---|---|---|

IMI | Internalization minimal inoculum | IMI is a virtual value extrapolated from the equation of the regression curve achieved by plotting Log MOI vs. Log (CFU). IMI corresponds to the lowest MOI required for the internalization of a single bacterium. The lower the value the higher the invasiveness of the strain. IMI lowest value, in our system, would correspond to 0.0001:1 MOI, i.e., a suspension containing a single bacterium for 10^{4} eukaryotic cells. Being inversely related to invasiveness, IMI could advantageously be used to express the resistance to phagocytosis. |

I1M | Internalization at 1:1 MOI inoculum | I1M is a virtual value, extrapolated from the equation of the regression curve achieved by plotting Log MOI vs. Log (CFU). I1M corresponds to the number of bacteria internalised when hypothetically exposing each eukaryotic cell to a single bacterium (i.e., when using a 1:1 MOI). Its value is directly proportional to the degree of invasiveness of the strain. |

LIMI | Log_{10} of the IMI value | LIMI is promptly obtained from the regression curve of Log MOI vs. Log (CFU). Its value, expected in the range from −3 to 4, is inversely proportional to the degree of invasiveness of the bacterial strain. LIMI exhibits a lower coefficient of variation with respect to IMI, PIB and I1M, and it more closely approaches a normal distribution. LIMI can be easily transformed to obtain the corresponding IMI value. |

LI1M | Log_{10} of the I1M value | LI1M is promptly obtained from the regression curve of Log MOI vs. Log (CFU). Its value, expected in the range from −4 to 3, is directly proportional to the degree of invasiveness of the bacterial strain. LI1M exhibits a lower coefficient of variation with respect to IMI, PIB and I1M, and it more closely approaches a normal distribution. It can be easily transformed to obtain the corresponding I1M value. |

Bacterium | PIB [Mean ± S.D. ^{1}/(C.V.)] | IMI [Mean ± S.D./(C.V.)] | I1M [Mean ± S.D./(C.V.)] | LIMI [Mean ± S.D./(C.V.)] | LI1M [Mean ± S.D./(C.V.)] |
---|---|---|---|---|---|

S. aureus (HI + LI) | 8.89 ± 8.88 (99.9%) | 0.97 ± 2.04 (211.1%) | 693.67 ± 696.23 (100.4) | −2.18 ± 1.61 (74.1%) | 2.08 ± 1.15 (74.3%) |

S. aureus (HI) | 11.43 ± 8.45 ^{h}(73.9%) | 0.0014 ± 0.0012 ^{l}(83.2%) | 891.79 ± 663.52 ^{h}(74.4%) | −2.97 ± 0.35 ^{a,b,c,d}(11.7%) | 2.85 ± 0.33 ^{a,b,c,d}(11.6%) |

S. aureus (LI) | 0.0029 ± 0.0016 (43.9%) | 4.34 ± 1.99 (45.7%) | 0.27 ± 0.12 (44.4%) | 0.61 ± 0.21 ^{a,f}(33.5%) | −0.60 ± 0.20 ^{a,f}(33.4%) |

S. epidermidis | 0.0010 ± 0.0005 (49.9%) | 13.2 ± 10.0 (76.1%) | 0.15 ± 0.09 (63.0%) | 1.02 ± 0.30 ^{b,e}(29.6%) | −0.92 ± 0.33 ^{b,e}(35.5%) |

S. lugdunensis | 0.00027 ± 0.00012 ^{l}(46.2%) | 46.6 ± 24.7 ^{h}(53.0%) | 0.032 ± 0.016 ^{l}(50.8%) | 1.63 ± 0.20 ^{c,e,f}(12.4%) | −1.54 ± 0.23 ^{c,e,f,g}(14.7) |

E. faecalis | 0.0011 ± 0.0011 (95%) | 9.2 ± 9.37 (101.5%) | 0.35 ± 0.15 (42.1%) | 0.81 ± 0.55 ^{d}(68.6%) | −0.47 ± 0.19 ^{d,g}(39.8%) |

^{1}Note that the mean of PIB and the respective S.D. values were calculated after averaging the many measurements performed per each strain at different MOI. S.D. and C.V. are therefore lower than what could be found with conventional single measurements that are based just on a MOI. The non-parametric Kruskal–Wallis test followed by Kruskal–Wallis ranking was used to analyse the means differences for PIB, IMI and I1M. The tied-p values for the three comparative analyses were respectively of 0.0009, 0.0009 and 0.0008. HI S. aureus strains diverged from the behaviour of the strains of the other species previously described as incompetent. Here, only the more detailed statistical analyses considering S. aureus split into the two HI and LI subcategories are shown (HI, highly internalising strains; LI, low internalising strains). The heterogeneous behaviour of S. aureus strains appeared distributed in two distinct clusters clearly causing a bimodal distribution. Logarithmic normalization enabled the use of post-hoc cell analysis by the Bonferroni/Dunn test. For LIMI and LI1M, superscript letters within the same column indicate the cross-comparisons statistically significant after performing the test.

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**MDPI and ACS Style**

Campoccia, D.; Montanaro, L.; Ravaioli, S.; Cangini, I.; Testoni, F.; Visai, L.; Arciola, C.R.
New Parameters to Quantitatively Express the Invasiveness of Bacterial Strains from Implant-Related Orthopaedic Infections into Osteoblast Cells. *Materials* **2018**, *11*, 550.
https://doi.org/10.3390/ma11040550

**AMA Style**

Campoccia D, Montanaro L, Ravaioli S, Cangini I, Testoni F, Visai L, Arciola CR.
New Parameters to Quantitatively Express the Invasiveness of Bacterial Strains from Implant-Related Orthopaedic Infections into Osteoblast Cells. *Materials*. 2018; 11(4):550.
https://doi.org/10.3390/ma11040550

**Chicago/Turabian Style**

Campoccia, Davide, Lucio Montanaro, Stefano Ravaioli, Ilaria Cangini, Francesca Testoni, Livia Visai, and Carla Renata Arciola.
2018. "New Parameters to Quantitatively Express the Invasiveness of Bacterial Strains from Implant-Related Orthopaedic Infections into Osteoblast Cells" *Materials* 11, no. 4: 550.
https://doi.org/10.3390/ma11040550