Five millennia of Bartonella quintana bacteraemia.

During the two World Wars, Bartonella quintana was responsible for trench fever and is now recognised as an agent of re-emerging infection. Many reports have indicated widespread B. quintana exposure since the 1990s. In order to evaluate its prevalence in ancient populations, we used real-time PCR to detect B. quintana DNA in 400 teeth collected from 145 individuals dating from the 1st to 19th centuries in nine archaeological sites, with the presence of negative controls. Fisher's exact test was used to compare the prevalence of B. quintana in civil and military populations. B. quintana DNA was confirmed in a total of 28/145 (19.3%) individuals, comprising 78 citizens and 67 soldiers, 20.1% and 17.9% of which were positive for B. quintana bacteraemia, respectively. This study analysed previous studies on these ancient samples and showed that the presence of B. quintana infection followed the course of time in human history; a total of 14/15 sites from five European countries had a positive prevalence. The positive rate in soldiers was higher than those of civilians, with 20% and 18.8%, respectively, in the 18th and 19th centuries, but the difference in frequency was not significant. These results confirmed the role of dental pulp in diagnosing B. quintana bacteraemia in ancient populations and showed the incidence of B. quintana in both civilians and soldiers.

Introduction

In June 1915, a British military doctor on the western front of the First World War reported the first case of recurrent fever in a soldier who presented with a headache, dizziness, and severe pain in the lower back and leg. Additional cases with the same clinical features were reported among soldiers in the trenches and it began to be referred to as “trench fever” [1]. The causative agent, Rickettsia quintana, was isolated from a patient in Mexico City by Vinson in 1961 and was reclassified in the genus Rochalimaea. In 1993, Rochalimaea merged with Bartonella [2, 3]. The vector for transmission is the human body louse (Pediculus humanus corporis), the gastrointestinal tract of which is colonised by B. quintana. It is shed in lice faeces, and penetrates the human body through damaged skin, entering the bloodstream [4, 5]. The typical cycle of fever occurred at five-day intervals (hence its other name, the “five-day fever”), resulting in prolonged disability so that affected soldiers were unfit for at least two months, many of them suffering from chronic fatigue [2]. Morbidity rates were not revealed by the authorities at the time, and no deaths were reported [2, 3]. The clinical manifestations of B. quintana infection range from asymptomatic to a severe, life-threatening infection. These include, in addition to trench fever, lymphadenopathy, bacillary angiomatosis, and chronic bacteraemia and endocarditis [5, 6]. In the absence of any appropriate treatment, the potential for relapse is due to the existence of an intraerythrocytic phase, as erythrocytes host bacteria during their life cycle [7]. Since the 1990s, B. quintana has been recognised as a re-emerging agent in homeless populations as a result of unsanitary living conditions [8-11].

The presence of trench fever can be traced back to the military in Europe before the First World War, when B. quintana DNA sequences were found in dental pulp from Napoleonic soldiers in Vilnius (1812) [12], and in bone from soldiers in Kassel (1813–814) [13]. Indeed, dental pulp with high vascularity has been the definitive key to diagnosing bacteraemia caused by microorganisms in paleomicrobiology [14]. Molecular biology has confirmed B. quintana bacteraemia occurred between 200 and 4,000 years ago, through the identification of B. quintana in dental pulp [15]. There has been no comprehensive study of the prevalence of the pathogen over the past two millennia in Europe. In order to perform such a study, we reviewed all paleomicrobiological data regarding B. quintana and combined this with the study of 400 additional specimens collected from 145 individuals over 20 centuries.

Materials and methods

Ancient human samples

Ancient teeth were collected from human remains in nine European burial sites by archaeologists and we were granted permission by the scientific managers of these sites to conduct microbiology studies. These samples are not restricted by any regulations in force and only require the authorisation of the scientific managers of these sites (all of whom have been acknowledged as co-authors) (S1 Text).

The specimen numbers used in this study are:

Besançon–France (1st– 4th): Number from 1 to 29.

Catacombs of St. Lucia–Italy (3rd - 6th): Number from 1 to 29.

San Basilio–Italy (4th– 6th): Number from 1 to 8.

Dueville–Italy (6th– 7th): Number from 1 to 15.

Remiremont–France (5th– 10th): Number from 1 to 45.

Amiens–France (18th– 19th): Number from 1 to 55.

Dax–France (1792–1833): Number from 1 to 110.

Kaliningrad–Russia (1812): Number from 1 to 30.

Sevastopol- Ukraine (1853–1856): Number from 1 to 79.

No permits were required for the described study and this study complied with all relevant regulations.

Following our current protocol for selecting and handling teeth, teeth were washed with sterile water and gradually dried, and the dental pulp was extracted using rotating disk instruments [16]. Total DNA was extracted using the phenol-chloroform protocol [17].

Molecular detection

Dental pulp was tested for B. quintana DNA using quantitative real-time PCR (qPCR) targeting the ITS gene using the following primers and probes: probe/6FAM-GCG CGC GCT TGA TAA GCG TG-TAMRA, forward/5’-GAT GCC GGG GAA GGT TT TC-3’, reverse/5’’ [18-20]. qPCR amplification was performed using the LightCycler® 480 Probes Master Kit according to the manufacturer’s recommendations (Roche Diagnostics, Meylan, France). Each well contained 10 μL of mix, 3 μL of sterile water, 0.5 μL of probe (20 μM), 0.5 μL of each primer (50 μM), 0.5 μL UDG, and 5 μL of extracted DNA. Amplification consisted of a two-minute incubation step at 50°C and an initial five-minute denaturation at 95°C, followed by 40 cycles of denaturation at 95°C for five seconds and hybridisation at 60°C for 30 seconds. Two negative controls (control of DNA extraction + mix and sterile water + mix) were placed every five samples in each plate. A sample was considered positive when the qPCR result was positive with a cycle number (Ct) lower than 40.

Fisher’s exact test

Fisher’s exact test was used to compare the prevalence of B. quintana infection in the civilian population and military population, and a p-value < 0.05 was considered statistically significant.

Results

A total of 400 ancient teeth from 145 individuals collected in nine European sites with different times of burial were analysed using qPCR. All negative controls were negative, and qPCR detected B. quintana DNA in all sites. The results indicated that a total of 78 civilians from six sites had 17.9% positivity, and 67 soldiers from three sites had 20.1% positivity. The positive detection at the Besançon site was the oldest, with data from the 1st– 4th centuries (Table 1). Seven of the 17 archaeological sites had data from the 18th– 19th centuries (Tables 1 and 2), where the prevalence of B. quintana DNA was 3/16 (18.8%) among civilians and 24/120 (20%) among soldiers; the difference in the prevalence was not significant (Table 3).

Table 1

Biomolecular results of B. quintana infection in ancient dental pulp.

Sites	Date	Positive teeth/total	Positive people/total	Number of teeth/person	Positive percentage	Population
Besançon—France	1st–4th	7/29	3/5	4–7	17.9% (14/78)	Civilian
Catacombs of St. Lucia—Italy	3rd–6th	1/29	1/28	1–2
San Basilio—Italy	4th–6th	1/8	1/6	1–2
Dueville—Italy	6th–7th	2/15	2/15	1
Remiremont—France	5th–10th	13/45	4/8	3–8
Amiens—France	18th–19th	4/55	3/16	3–4
Dax—France	1792–1833	11/110	4/9	9–14	20.1% (14/67)	Military
Kaliningrad—Russia	1812	2/30	2/30	1
Sevastopol- Ukraine	1853–1856	14/79	8/28	1–5

Table 2

Bartonella quintana detection in ancient specimens from previous studies.

Sites	Date	Specimens	Methods	Positive number of specimens/total	Positive number of people/total	Population	Ref.
Peyraoutes—France	2000BCE	Dental pulp	Suicide PCR (groEL, hbpE genes)	1/6	1/3	-	[21]
Roaix—France	2200BCE–2100BCE			0	0/3
Bondy—France	11th–15th		Real time PCR (ITS gene)	3/14	3/5	Civilian	[18]
Venice—Italy	15th–16th			5/173	-	Civilian	[19]
Douai—France	18th			1/40	-	Military	[20]
Vilnius—Lithuania	1812		Suicide PCR (hbpE, htrA genes)	7/72	7/35	Military	[12]
		Lice	PCR standard (hbpE gene)
Kassel -Germany	1813–1814	Bone	PCR standard (hbpE gene)	3/18	3/18	Military	[13]
Namur—Belgium	14th	Coprolite	Metagenomics				[22]

(-): Not mentioned

Table 3

Comparison of infected populations of 18th–19th centuries.

Population	Total	Positive number	Percentage	p
Civilians	16	3	18.8%	> 0.05
Soldiers	120	24	20%

Discussion

The results reported here were authenticated by the fact that we chose ancient teeth, preferentially monoradicular teeth with a close apex, no traumatic lesions, and an absence of dental caries that helped minimise any risk of external contamination [16]. A positive control was not used in the PCR experiments because it could be a source of contamination; the negative controls remained negative [18]. To screen a total of 400 teeth, we used real-time PCR as previously described to identify B. quintana [18-20], and in this work, each experimental step was performed in different rooms of a new building, where these gene sequences had not previously been used. The number of teeth per individual was variable in each burial site due to availability, and we used as many teeth per individual as possible to increase the chance of detection. Indeed, the first report of using dental pulp for ancient septicaemia diagnosis indicated that the number of teeth per individual infected with Yersinia pestis was 1/4 teeth, 1/2 teeth, and 3/3 teeth [14]. Furthermore, two teeth from one individual reported that the first was infected with Y. pestis and the second had co-infection of Y. pestis and B. quintana [18]. Accordingly, in several instances, co-detection of B. quintana with another deadly pathogen has been reported, such as Yersinia pestis, Rickettsia prowazekii, indicating that the detection of B. quintana does not preclude that of other pathogens in any archaeological site [12, 18, 23].

In 1915, the history of trench fever was marked by the report of new clinical features from soldiers involved in trench warfare [2, 3]. In 2005, authentic evidence showed that B. quintana had caused bacteraemia in humans for more than 4,000 years, through analysis of DNA from dental pulp collected from Peyraoutes in France [21]. This pathogen was also later identified in this specimen from different burial sites, including Venice (15th–16th centuries) [19], Douai (18th century) [20], Vilnius (1812) [12] and Bondy (11th–15th centuries) where an individual had coinfection with Y. pestis [18]. In addition to dental pulp, B. quintana was detected in ancient bone (1813–1814) [13] and coprolites (14th) century [22]. The bacterial confirmation in ancient pulp indicates that individuals had bacteraemia before their death, but it may not have been the cause of death, supporting the fact that no deaths have been recognised as being caused by trench fever [2, 5, 24]. A survey of 930 homeless people in Marseille revealed that 5.3% were blood culture positive for B. quintana [8], and asymptomatic chronic bacteraemia could be maintained for 78 weeks [25]. In 2004, B. quintana was found in the dental pulp of a homeless patient who had had bacteraemia in the previous six months [26].

Most of the reports of B. quintana in dental pulp come from our laboratory, with nearly half of burial sites being located in the 18th–19th centuries; 7/14 sites were in France, 4/14 were in Italy, and the rest came from Ukraine, Russia, and Lithuania. This can be explained by the availability of samples in these periods, and the conditions in which our research team in France operate made it highly favourable to receiving these teeth from archaeological centres. The wide geographical and temporal distribution suggests that this bacterial infection was common in historic European populations. In the medical literature, the “five-day fever” and the Moldavia fever in the 19th century had clinical signs similar to trench fever but laboratory techniques to confirm this were lacking at that time [27].

Millions of soldiers were contaminated around the world during the two World Wars. After the end of each war, however, incidence dropped dramatically but still sporadically persisted in some countries [5, 24]. It was noted that Napoleonic soldiers buried in Vilnius, Lithuania (1812) were exposed to body lice containing B. quintana, and approximately 20% and 8.6% of soldiers were infected with B. quintana and R. prowazekii, respectively [12]. An investigation of ancient bones from 18 soldiers from a mass grave inhumed in the winter of 1813–14 in Kassel, Germany, revealed that 16.7% of the soldiers had the infection [13]. In our study, only 6.7% of the soldiers from the Kaliningrad site tested positive, in contrast to the higher positive rates from Dax and Sevastopol. This can be explained by the use of only one tooth per individual for the Kaliningrad samples. The catacombs in St. Lucia, which represented the ancient Christian monument of the Late Roman period [28], had the similarity of also using one tooth, with the lowest positive proportion (3.6%). During wartime, soldiers were generally crowded close together in unsanitary conditions for prolonged periods of time. Moreover, despite knowing the role that lice played in transmission and their presence in clothes, there were no effective methods of disinfection available [2, 3].

In order to ensure we were working with a uniform time period, we chose the 18th–19th centuries, which had a greater number of both populations. The incidence in civilians and soldiers was 18.8% and 20%, respectively, and there was no significant difference. This suggests that this bacterial infection was relatively common throughout human society, without any detectable difference in lice infestation and clothing hygiene, although the sample size of citizens was small (18 citizens), which could affect the comparison. The epidemiology of trench fever involves interhuman transmission via vectors, such as body louse infection, or other transmission that may involve contact with reservoirs, insects bites [29], cat bites [30] or others that have yet to be defined and need further elucidation. Humans are not the only reservoirs of B. quintana; it is also found in cat fleas [31] and monkey fleas [32], and some studies have indicated other animals as known reservoirs through the detection of B. quintana in domestic cats [33], dogs [34], rhesus macaques [35, 36], and Japanese macaques [37]. Following an experimental study on cat fleas, it was shown that this pathogen was absorbed via the gastrointestinal tract and released into faeces [38]. Another study showed that the Pedicinus obtusus louse was postulated as an efficient vector of transmission between rhesus macaques [36]. Homelessness was defined as a high-risk factor for B. quintana infection as a consequence of inadequate hygiene [8, 10, 11, 25]; however, some subpopulations have relatively high exposure rates, such as blood donors, with a rate of between 27% and 51% [9, 39, 40], and healthy people, with a rate of between 11.2% and 25% [40-43]. There is no evidence to show that these people lived in unhygienic living conditions, such as being homeless, or that they had come into contact with body lice, suggesting the existence of some underestimated factors that cause a bacterial infection. The new findings on transmission, transmitted vectors, reservoirs, and widely infected populations contribute to the understanding of the infection from the past to the present; paleomicrobiological studies as this one helping to clarify complex host-pathogen interactions in past human populations, as previously reported [44, 45].

Conclusions

Dental pulp is conducive to the investigation of B. quintana bacteraemia in ancient populations, and it is relatively easy to collect and is well protected inside the tooth. Previous studies analysed in this study showed the presence of B. quintana infection 4,000 years ago and between the 1st and 19th centuries. Soldiers are considered the main target of this bacterial infection in human history, but in this study, we saw no significant difference between civilians and soldiers during the 18th and19th centuries.

Nine European archeological sites.

(DOCX)

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23 Apr 2020

PONE-D-20-07460

Five millennia of Bartonella quintana bacteremia.

PLOS ONE

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Reviewer #1: I have genuinely appreciated the spirit of the paper and the original data presented by the authors of this manuscript.

The topic of B. quintana infection is interesting and the genetic data derived from ancient samples can help science reconstruct the evolutionary history of this pathogen, its interaction with the human species and its global impact on humankind's health.

However, there are some issues that the authors should carefully consider and work on:

1. the English of the manuscript is generally week, with several expressions and collocations typically seen in other languages. In addition, the names of pathogens are not consistently written in italics throughout the manuscript. The manuscript should be read by a native speaker;

2- the tone of the manuscript is often too assertive e.g. "the results reported here are authentic";

3- the general clinical aspects of B. quintana infection should also be presented in the "Introduction" section, as much as the fact that presence of the pathogen does not implicate a severe clinical manifestation. Furthermore, the historical part on the first description of trench fever should be discussed more at length, with a focus on the overall impact on soldiers' health, as much as its interplay with the subsequent Spanish flu pandemic (they are correct in touching co-morbidities later on in the manuscript);

4- talking of comorbidities, the authors should endeavour to speculate more in depth on the potential health consequences on the analyzed individuals;

5- the comparison between infection in soldiers and civilians is interesting but mostly "artificial" because this disease was considered a "military disease" at the very beginning of its recorded history only due to the (special) circumstances under which it was originally described, but there is no real reason to think that poor hygiene conditions are an exclusive characteristic of military life. Way more interesting would be to examine the demographic information for each site, e.g. male/female ratio, distribution according to age range, living standards, diet, detected co-morbidities;

6- in the article, the authors also touch upon the topic of animal infection and zoonotic transmission to the human species, which is indeed a very good point. Further details should be given for the ancient populations analyzed in this study;

7- with reference to the St. Lucia Catacomb (Syracuse, Sicily), from the scientific perspective and that of science communication, I find it controversial that on 8th April 2019 an article (https://www.lasicilia.it/gallery/sicilians/234328/davide-tanasi-l-archeologo-che-dagli-stati-uniti-svela-la-sicilia-antica.html) appeared in the Italian-language press where directly quoted statements were made about the presence in two individuals of "febbre del legionario" - possibly referred to "Legionnaires' disease"?, a completely different disease caused by Legionella pneumophila - while in this manuscript it is written that only 1/28 citizens tested positive.

Are we talking about the same sample(s) presented in this paper or something published elsewhere/unpublished?

If we are talking about the same sample(s), has something changed in the lab data in the meantime? Interestingly, in that press report, a correlation was made between poor diet and disease (exactly what I mentioned in point #5). Why has this not been investigated further in the official scientific publication?

Reviewer #2: It is an interesting manuscript which shows, however, some relevant lacunae.

A. In the abstract the last sentence is not clear: are the Author/s talking about their findings or findings in this area in general? Please clarify this point.

B. Insufficient demographic data are provided for each site in the supplementary material (6 out of 9 sites have no anthropological data). I also suggest to use this data in the context of a lengthier discussion in the main manuscript.

C. The manuscript lacks information about the skeletons and teeth that were examined: for example table 1 should also contain more details about the samples, not only how many and whether they tested positive. In order to better assess the quality of the analyzed sample, I would like to know which specific tooth, from which side, if it is monoradicular or pluriradicular and also the sex and age at death of the skeletons considered. Or you can provide another table with this information. This nice genetic result, to achieve a higher value, should be better related to a proper and exhaustive demographic or, at least when not possible, anthropological discussion.

An addition like this would increase the quality of the manuscript.

D. Do the Author/s have any data on the dietary conditions of these ancient individuals? Literary or archaeological or isotope analyses.

E. In the discussion (page 5), the Author/s write "free of traumatic lesions": can the Author/s better clarify this point? Are the Author/s talking about the absence of pathologies like caries or calculus and also tooth fractures?

F. The English of the manuscript shows some impurities: a check by a native speaker would be a good option.

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24 Jun 2020

Dear Editor,

Please find enclosed the revised version of our manuscript PONE-D-20-07460 entitled “Five millennia of Bartonella quintana bacteremia” along with the answers of the authors to the reviewers’remarks.

Please note that all the authors agreed on the addition of David Peressinotto, and Coralie Demangeot as additional co-authors which now contribution helped to answer some reviewers’ comments.

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'all the teeth studied, we were provided by their scientific managers having the full

liberty to conducted microbiology studies. This kind of sample is not restricted by any

regulation in force and only requires the authorization of the scientific manager (all

granted as co-authors)'

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Authors’answer: The text has been corrected accordingly and the appropriate section has been completed in the website.

6. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 1-3 in your text; if accepted, production will need this reference to link the reader to the Table.

Authors’answer: The authors corrected mislabelling of Tables, all the three Tables are now cited in the text (Lines 121-125).

7. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files

Authors’answer: Corrected accordingly

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Authors’answer: The authors corrected mislabelling of Tables, there are NO supplementary Tables.

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Authors’answer: Corrected accordingly, line 78 in the text (S1 Text) and Supporting Information, line 323-324

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: I have genuinely appreciated the spirit of the paper and the original data presented by the authors of this manuscript.

The topic of B. quintana infection is interesting and the genetic data derived from ancient samples can help science reconstruct the evolutionary history of this pathogen, its interaction with the human species and its global impact on humankind's health.

However, there are some issues that the authors should carefully consider and work on:

1. the English of the manuscript is generally week, with several expressions and collocations typically seen in other languages. In addition, the names of pathogens are not consistently written in italics throughout the manuscript. The manuscript should be read by a native speaker;

Authors’answer: The revised version of the manuscript has been corrected by professionals (Certificate enclosed).

2- the tone of the manuscript is often too assertive e.g. "the results reported here are authentic";

Authors’answer: The revised version of the manuscript has been corrected by professionals who took this appreciation (which is just incomprehensible for French authors) into consideration (Certificate enclosed).

3- the general clinical aspects of B. quintana infection should also be presented in the "Introduction" section, as much as the fact that presence of the pathogen does not implicate a severe clinical manifestation. Furthermore, the historical part on the first description of trench fever should be discussed more at length, with a focus on the overall impact on soldiers' health, as much as its interplay with the subsequent Spanish flu pandemic (they are correct in touching co-morbidities later on in the manuscript);

Authors’answer:

- The clinical manifestations of infection presented in the Introduction section are now expanded as proposed by the reviewer (Lines 55-58).

- The authors do not agree with the following remark of the reviewer that B. quintana infection is a mild one. The authors are now reporting on mortality rates which clearly indicate that B. quintana infection is indeed a life-threatening infection (Lines 52-55).

- The authors carefully checked that no report issued regarding B. quintana and Spanish flu (see reference [2,3], line 211-213).

4- talking of comorbidities, the authors should endeavour to speculate more in depth on the potential health consequences on the analyzed individuals;

Authors’answer: The authors thanks the reviewer to give the opportunity to discuss this interesting aspect (Lines 139-142).

5- the comparison between infection in soldiers and civilians is interesting but mostly "artificial" because this disease was considered a "military disease" at the very beginning of its recorded history only due to the (special) circumstances under which it was originally described, but there is no real reason to think that poor hygiene conditions are an exclusive characteristic of military life. Way more interesting would be to examine the demographic information for each site, e.g. male/female ratio, distribution according to age range, living standards, diet, detected co-morbidities;

Authors’answer: Indeed, one output of the present study was to demonstrate an absence of significant difference between civil and military populations; hence suggesting the absence of differences in terms of cloth hygiene (Lines 180-182).

6- in the article, the authors also touch upon the topic of animal infection and zoonotic transmission to the human species, which is indeed a very good point. Further details should be given for the ancient populations analyzed in this study;

Authors’answer: The authors just not have any data regarding contacts with animals in any of the buried populations here reported.

7- with reference to the St. Lucia Catacomb (Syracuse, Sicily), from the scientific perspective and that of science communication, I find it controversial that on 8th April 2019 an article (https://www.lasicilia.it/gallery/sicilians/234328/davide-tanasi-l-archeologo-che-dagli-stati-uniti-svela-la-sicilia-antica.html) appeared in the Italian-language press where directly quoted statements were made about the presence in two individuals of "febbre del legionario" - possibly referred to "Legionnaires' disease"?, a completely different disease caused by Legionella pneumophila - while in this manuscript it is written that only 1/28 citizens tested positive.

Are we talking about the same sample(s) presented in this paper or something published elsewhere/unpublished?

If we are talking about the same sample(s), has something changed in the lab data in the meantime? Interestingly, in that press report, a correlation was made between poor diet and disease (exactly what I mentioned in point #5). Why has this not been investigated further in the official scientific publication?

Authors’answer:

- The reviewer is perfectly right to point to the possibility of co-infection with Bartonella quintana in ancient populations, and this interesting point is now emphasized in the revised version of the manuscript (Lines 139-142).

- Then, to the best of the authors knowledge, co-infections with Bartonella quintana are with other lice-borne pathogens including Yersinia pestis, Rickettsia prowazekii; this point is now clarified in the revised version of the manuscript (Lines 139-142).

- Then, to the best of the authors knowledge, Legionella pneumophila is NOT known to be a lice-borne pathogen; but rather a water-borne pathogen currently transmitted by aerosols of contaminated water.

- Therefore, there was no particular reason to test for the hypothesis of Legionella pneumophila rather than any other hypothesis.

- At last, the “La Sicilia” paper referred to by the reviewer, is clearly grey literature which cannot be incorporated nor discussed as a reference in the present work to be published in PLoS ONE.

- The authors did not test for Legionella pneumophila but rather for Bartonella quintana; the 1/28 individual in Table 1 is referring to Bartonella quintana, not at all to Legionella pneumophila.

Reviewer #2: It is an interesting manuscript which shows, however, some relevant lacunae.

A. In the abstract the last sentence is not clear: are the Author/s talking about their findings or findings in this area in general? Please clarify this point.

Authors’answer: The reviewer is perfectly right and this sentence was deleted from the revised version.

B. Insufficient demographic data are provided for each site in the supplementary material (6 out of 9 sites have no anthropological data). I also suggest to use this data in the context of a lengthier discussion in the main manuscript.

Authors’answer: The reviewer is right but no further demographic data were available in the reasonable time for revising this manuscript.

C. The manuscript lacks information about the skeletons and teeth that were examined: for example table 1 should also contain more details about the samples, not only how many and whether they tested positive. In order to better assess the quality of the analyzed sample, I would like to know which specific tooth, from which side, if it is monoradicular or pluriradicular and also the sex and age at death of the skeletons considered. Or you can provide another table with this information. This nice genetic result, to achieve a higher value, should be better related to a proper and exhaustive demographic or, at least when not possible, anthropological discussion.

An addition like this would increase the quality of the manuscript.

Authors’answer: The reviewer is right but no further demographic data were available in the reasonable time for revising this manuscript.

D. Do the Author/s have any data on the dietary conditions of these ancient individuals? Literary or archaeological or isotope analyses.

Authors’answer: The reviewer is right but no further demographic data were available in the reasonable time for revising this manuscript.

E. In the discussion (page 5), the Author/s write "free of traumatic lesions": can the Author/s better clarify this point? Are the Author/s talking about the absence of pathologies like caries or calculus and also tooth fractures?

Authors’answer: The reviewer is perfectly right, this point is clarified both in the Discussion section (Lines 128-130).

F. The English of the manuscript shows some impurities: a check by a native speaker would be a good option.

Authors’answer: The revised version of the manuscript has been corrected by professionals (Certificate enclosed).

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

As the authors answered all the Editor and Reviewers’ remarks and corrected the manuscript accordingly, they hope that this revised version will be accepted for publication in PLoS ONE.

Sincerely,

Dr. Gérard ABOUDHARAM,

Corresponding author.

Submitted filename: Plos One answers.docx

Click here for additional data file.

11 Aug 2020

PONE-D-20-07460R1

Five millennia of Bartonella quintana bacteremia.

PLOS ONE

Dear Dr. Aboudharam,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the minor point highlighted by one referee that are reported below .

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: N/A

Reviewer #2: N/A

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This new version of the manuscript is well-researched and presented in a better form. In my opinion, the authors have made good amendments.

The "La Sicilia" reference be may grey literature but I am very skeptical about going to the media with certain claims and then issuing something different in a scientific paper, but thanks for clarifying that.

I would only recommend adding a couple of general references when you discuss the pathogen-population interaction:

1) Bos KI, Kühnert D, Herbig A, et al. Paleomicrobiology: Diagnosis and Evolution of Ancient Pathogens. Annu Rev Microbiol. 2019;73:639-666.

2) Rühli FJ, Galassi FM, Haeusler M. Palaeopathology: Current challenges and medical impact. Clin Anat. 2016;29(7):816-822.

Reviewer #2: Edits made in an orderly and clear way. I think the authors have answered the main critical points of the previous version of this manuscript.

**********

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18 Aug 2020

Dear Editor,

Please find enclosed the second revised version of the manuscript PONE-D-20-07460 entitled: “Five millennia of Bartonella quintana bacteremia” along with the answers of the authors to the reviewers ‘ comments.

Reviewers' comments:

Reviewer #1: This new version of the manuscript is well-researched and presented in a better form. In my opinion, the authors have made good amendments.

The "La Sicilia" reference be may grey literature but I am very skeptical about going to the media with certain claims and then issuing something different in a scientific paper, but thanks for clarifying that.

Authors’answer: The authors acknowledge this positive remark.

I would only recommend adding a couple of general references when you discuss the pathogen-population interaction:

1) Bos KI, Kühnert D, Herbig A, et al. Paleomicrobiology: Diagnosis and Evolution of Ancient Pathogens. Annu Rev Microbiol. 2019;73:639-666.

2) Rühli FJ, Galassi FM, Haeusler M. Palaeopathology: Current challenges and medical impact. Clin Anat. 2016;29(7):816-822.

Authors’answer: The reviewer is perfectly suggesting to broaden the Discussion section, these two references are now cited in the Discussion section (Lines 213-215), and listed as new references 44 and 45, accordingly.

Reviewer #2: Edits made in an orderly and clear way. I think the authors have answered the main critical points of the previous version of this manuscript.

Authors’answer: The authors acknowledge this positive remark.

As the authors answered the Reviewer n°1 remark and corrected the manuscript accordingly, the authors hope that this second revised version will be accepted for publication,

Sincerely

Gérard ABOUDHARAM,

Corresponding author.

Submitted filename: Rebuttal Letter.docx

Click here for additional data file.

9 Sep 2020

Five millennia of Bartonella quintana bacteremia.

PONE-D-20-07460R2

Dear Dr. Aboudharam,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

David Caramelli, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

7 Oct 2020

PONE-D-20-07460R2

Five millennia of Bartonella quintana bacteraemia.

Dear Dr. Aboudharam:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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PLOS ONE