Incidence of an Intracellular Multiplication Niche among Acinetobacter baumannii Clinical Isolates

ABSTRACT The spread of antibiotic-resistant Acinetobacter baumannii poses a significant threat to public health worldwide. This nosocomial bacterial pathogen can be associated with life-threatening infections, particularly in intensive care units. A. baumannii is mainly described as an extracellular pathogen with restricted survival within cells. This study shows that a subset of A. baumannii clinical isolates extensively multiply within nonphagocytic immortalized and primary cells without the induction of apoptosis and with bacterial clusters visible up to 48 h after infection. This phenotype was observed for the A. baumannii C4 strain associated with high mortality in a hospital outbreak and the A. baumannii ABC141 strain, which was isolated from the skin but was found to be hyperinvasive. Intracellular multiplication of these A. baumannii strains occurred within spacious single membrane-bound vacuoles, labeled with the lysosomal associate membrane protein (LAMP1). However, these compartments excluded lysotracker, an indicator of acidic pH, suggesting that A. baumannii can divert its trafficking away from the lysosomal degradative pathway. These compartments were also devoid of autophagy features. A high-content microscopy screen of 43 additional A. baumannii clinical isolates highlighted various phenotypes, and (i) the majority of isolates remained extracellular, (ii) a significant proportion was capable of invasion and limited persistence, and (iii) three more isolates efficiently multiplied within LAMP1-positive vacuoles, one of which was also hyperinvasive. These data identify an intracellular niche for specific A. baumannii clinical isolates that enables extensive multiplication in an environment protected from host immune responses and out of reach of many antibiotics. IMPORTANCE Multidrug-resistant Acinetobacter baumannii isolates are associated with significant morbidity and mortality in hospitals worldwide. Understanding their pathogenicity is critical for improving therapeutic management. Although A. baumannii can steadily adhere to surfaces and host cells, most bacteria remain extracellular. Recent studies have shown that a small proportion of bacteria can invade cells but present limited survival. We have found that some A. baumannii clinical isolates can establish a specialized intracellular niche that sustains extensive intracellular multiplication for a prolonged time without induction of cell death. We propose that this intracellular compartment allows A. baumannii to escape the cell’s normal degradative pathway, protecting bacteria from host immune responses and potentially hindering antibiotic accessibility. This may contribute to A. baumannii persistence, relapsing infections, and enhanced mortality in susceptible patients. A high-content microscopy-based screen confirmed that this pathogenicity trait is present in other clinical A. baumannii isolates. There is an urgent need for new antibiotics or alternative antimicrobial approaches, particularly to combat carbapenem-resistant A. baumannii. The discovery of an intracellular niche for this pathogen, as well as hyperinvasive isolates, may help guide the development of antimicrobial therapies and diagnostics in the future.

Rubia and coworkers report an exciting finding: the invasion and intracellular survival of A. baumannii in non phagocytic cells cells. An important strength of the study is the test of a significant number of clinical strains to demonstrate how widespread the phenotype it is. This work will catalyze significant research by other groups, and may have clinical implications as some of treatments used in the clinic do not target intracellular bacteria.
Major comments. 1. Fig 1D. The limited scope of the results (only one cytokine and not assessment of the activation of signalling pathways) makes necessary for the authors to tone down the comparison between strains. This reviewer urges authors to just indicate that the strains do elicit IL6, and that the levels increase over time. And that this result is not related to the number of intracellualr bacteria. In fact, this is the interesting observation.
2. An interesting observation across the manuscript is the apparent localization of the ACV near the nucleus. Could the authors quantify this? 3. A549 cells are notoriously robust cells. This reviewer will urge authors to test toxicity in other cell types using LDH released (or similar test). 4. Although it is used in this manner in papers, LAMP1 cannot be regarded as a lysosomal marker. These comments need to be remove from the text, and just refer to a late endosomal marker. 5. It would be appropriate to test the colocalization of the ACV with a lysosomal marker over time. 6. Lamp1, and lysotracker colocalization with the ACV should be assessed over time. 7. It would have been interesting to get a sense of the cellular elements implicated on A. baumannii engulfment (actin cytoskeleton, microtubules, PI3K,...) Reviewer #2 (Comments for the Author): In the manuscript by Rubio, et al., the authors investigate whether a subset of Acinetobacter baumannii isolates are able to enter and grow within normally nonphagocytic cells. The provide evidence that an isolate thought to be of increased virulence, C4, enters and grows within cells. Entry and growth occur without attendant death, and was also observed with the isolate ABC141, which appeared to be hyperinvasive. The compartment surrounding bacterial compartments was LAMP1-positive but appeared to be nonacidic. Similarly, there was no evidence for autophagy, in contrast with previous reports regarding A. baumannii. In a screen of over 40 isolates coming from various lineages showed intracellular multiplication of 4 of them and two that were hyperinvasive.
The manuscript is an interesting contribution to the Acinetobacter literature. My main issues with the manuscript are that nonstandard assays are used to measure and compare adhesion to uptake, and there is no real negative control to compare the data to. For instance, I believe that in Fig. 1B, the authors are challenging cells at an MOI =100, and they are seeing about 0.2% cell associated bacteria based on viable counts (this should be made clear). If they did the experiment with just E. coli K12 that is nonpiliated (a standard nonadhesive control) is this significantly different, given those high MOI conditions? In addition, standard uptake assays involve either differential antibody staining (before and after permeabilization of fixed cells) or aminoglycoside survival after uptake. The latter they may have performed, since they mentioned that in fluorescent microscopy they incubated cells with apramycin or tobramycin, but it is not clear that all the bacteria were sensitive to these antibiotics. The authors should also discuss whether there is any evidence in animal models for bacteria residing in nonphagocytic cells. Its possible this might occur in the urinary tract.
Detailed comments: 1. Fig. 1A: The authors state that C4 is significantly more virulent than AB17978, but it is impossible to evaluate this statement given the nature of the experiment. Is there some statistical test to show this is actually significant? 2. Fig. 1B: Please state more clearly how this experiment is done and that the quantitation is performed differently from Fig.2B. It took considerable time to figure out whether the difference was due to assay measures or cell line differences. 3. Fig. 1b Fig. 2B and throughout: A standard measure of uptake should be used, as described in the general comments (antibody protection or aminoglycoside protection). 6. Figure 2B, hyperinvasion of ABC141 and other strains: from the Staphylococcus literature, hyper-cell association can be attributed to loss of capsule. Particularly with ABC141, and the hyperinvasive strains in Fig. 5, is this due to absence of capsule production? For instance, with ABC141, its possible that capsule is only produced in post-exponential phase, since uptake only occurs during exponential growth.

Dear Editors,
Please find bellow our point-by-point answer to the reviewers. We have also noticed a mistake in the Figure 5 for one of the strains (ABC020) from the screen that is capable of intracellular multiplication. This is now corrected. In addition, small edits were made throughout the text to correct for typos and misuse of the word "strain" vs "isolate".

Reviewer #1
1. Fig 1D. The limited scope of the results (only one cytokine and not assessment of the activation of signalling pathways) makes necessary for the authors to tone down the comparison between strains. This reviewer urges authors to just indicate that the strains do elicit IL6, and that the levels increase over time. And that this result is not related to the number of intracellualr bacteria. In fact, this is the interesting observation. The text has been modified accordingly.

An interesting observation across the manuscript is the apparent localization of the ACV near the nucleus. Could the authors quantify this?
This is indeed the case for a significant proportion of infected cells presenting large clusters but seems to only be occurring at 24h. We are currently investigating if this location corresponds to the MTOC and if vacuole localization is dependent on microtubules. However, as this is still very preliminary and requires live-imaging for confirmation we prefer not to include this aspect in the manuscript.

A549 cells are notoriously robust cells. This reviewer will urge authors to test toxicity in other cell types using LDH released (or similar test).
We have expanded our cytotoxicity analysis as recommended. We have now analysed both A549 and EA.hy 926 endothelial cells by measuring LDH release but also caspase activation to enable single cells analysis (of infected cells only), at both 24 and 48h post-infection. The results are now included in Figures 2G-J and the text modified.

4.
Although it is used in this manner in papers, LAMP1 cannot be regarded as a lysosomal marker. These comments need to be remove from the text, and just refer to a late endosomal marker. The text was modified accordingly.

It would be appropriate to test the colocalization of the ACV with a lysosomal marker over time.
These data are now included in Figure 3D and the legends and text modified.

Lamp1, and lysotracker colocalization with the ACV should be assessed over time.
These data can be found in Figure 3D and the legends and text modified. A. baumannii engulfment (actin cytoskeleton,microtubules,PI3K,...) In this manuscript we did not focus on entry as there are several reports implicating actin and microtubules in entry of different A. baumannii strains into non-phagocytic cells (for example, Choi et al 2008). We are currently constructing fluorescent strains to extend these studies for the hyperinvasive strain ABC141.

Reviewer #2:
My main issues with the manuscript are that nonstandard assays are used to measure and compare adhesion to uptake, and there is no real negative control to compare the data to. For instance, I believe that in Fig. 1B, the authors are challenging cells at an MOI =100, and they are seeing about 0.2% cell associated bacteria based on viable counts (this should be made clear). If they did the experiment with just E. coli K12 that is nonpiliated (a standard nonadhesive control) is this significantly different, given those high MOI conditions? We understand the confusion and have now clarified the text. The experiment presented in Figure 1B   In addition, standard uptake assays involve either differential antibody staining (before and after permeabilization of fixed cells) or aminoglycoside survival after uptake. The latter they may have performed, since they mentioned that in fluorescent microscopy they incubated cells with apramycin or tobramycin, but it is not clear that all the bacteria were sensitive to these antibiotics.
To quantify the percentage of cells with intracellular bacteria we used microscopy analysis of cells labeled with phalloidin, allowing to establish intracellular location in relation to the actin cytoskeleton. This is now clarified in the text. We have carried out the suggested experiment, with differential labeling and the results are now included in Figure 2C. We do not feel CFU counts for intracellular bacteria are adapted as they do not allow for single cell analysis, which is now the gold-standard in the field. For example, they do not allow distinguishing a few heavily infected cells from many cells with only a few bacteria.
The authors should also discuss whether there is any evidence in animal models for bacteria residing in nonphagocytic cells. Its possible this might occur in the urinary tract. As probably this reviewer is aware from his/her suggestion, beautiful unpublished work from Hultgren's lab suggests this is indeed the case, a reservoir of replicating intracellular bacteria can be found in epithelial cells of the urinary tract in a mouse model of infection. We believe that it is not for us to carry out these experiments and compete with our colleagues and we will await the publication of their results.
Detailed comments: 1. Fig. 1A: The authors state that C4 is significantly more virulent than AB17978, but it is impossible to evaluate this statement given the nature of the experiment. Is there some statistical test to show this is actually significant?
The statistical analysis (log-rank test) was originally referred to in the Figure legend. We have now added the information to the Figure P= ****. Fig. 1B: Please state more clearly how this experiment is done and that the quantitation is performed differently from Fig.2B. It took considerable time to figure out whether the difference was due to assay measures or cell line differences.

2.
We have now modified the text to explain that Fig 1B corresponds to CFU counts and Fig 2B  microscopy counts. Fig. 1b, 2B. You really need E. coli K12 as a negative control here.

3.
The adhesion of A. baumannii is well established in the field. E. coli K12 showed no adhesion ( Figure A of this letter) and was therefore not an ideal control for invasion. Instead we have included A. baylyi, which shows equivalent adhesion to 17978 but for which we do not see any invasion (Fig. 2C). Fig. 1B to 2B: Why is it that there more C4 associated in Fig. 2B than 17978, but the number is reversed in Fig. 1B?

Comparing
In Figure 1B the difference between 17978 and C4 is not statistically significant. This is also the case for Figure 2B, as indicated in the figures. Fig. 2B and throughout: A standard measure of uptake should be used, as described in the general comments (antibody protection or aminoglycoside protection). Aminoglycoside protection assays have been frequently used in the past and might be useful in some circumstances. However, besides technical issues, they are less informative than single cell analysis techniques. E.g. one can have a single cell with 200 bacteria or 200 cells with 1 bacterium each and obtain the same CFU counts; yet the result is very different. Single cell analysis by microscopy or image-coupled cytometry are more precise methods. We have chosen a microscopy-based approach in this study. Figure 2B, hyperinvasion of ABC141 and other strains: from the Staphylococcus literature, hyper-cell association can be attributed to loss of capsule. Particularly with ABC141, and the hyperinvasive strains in Fig. 5, is this due to absence of capsule production? For instance, with ABC141, its possible that capsule is only produced in post-exponential phase, since uptake only occurs during exponential growth. This is an excellent comment and something we are starting to investigate. As we are not experts in capsule biology we are establishing the appropriate collaborations to do so. This is however, in our opinion, beyond the scope of this manuscript. Fig. 5: AB17978 is listed as a urine isolate. Just want to make sure this is correct. Apologies for the mistake. This has now been corrected.

Very minor point: I am curious why the authors chose clonal lineage analysis rather than MLST
We have performed several large global epidemiological studies with Acinetobacter baumannii, and have found that there are nine distinct lineages that are widely distributed throughout the world, i.e. they are international. Sequence types are good to cluster isolates, but there are two competing schemes, and so two different nomenclatures. Because of the international makeup of our isolates, we prefer to group them using the international clones nomenclature. Fig. 5 is very useful, but there are issues with some of these assays and the antibotics used to kill extracellular bacteria. Are all of these bacteria sensitive to the antibiotics used (tobramycin or apramycin)?

9.
The antibiotic data sensitivity is now included as Supplementary Fig1. Thank you for submitting your manuscript to mSystems. We have completed our review and I am pleased to inform you that, in principle, we expect to accept it for publication in mSystems. However, acceptance will not be final until you have adequately addressed the reviewer comments.
The reviewers and editor believe that the manuscript is much improved. However, the authors are asked to address the outstanding concern of reviewer 2 that it is unexpected that no adhesion of the control strain would be observed.
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