Associated Bacterial Coinfections in COVID-19-Positive Patients

Background and Objectives: The aim of this study was to identify specific rhino- and oropharyngeal microbiological pathogens as well as associated comorbidities that favor SARS-CoV-2 infection and corelate them. Materials and Methods: This prospective clinical study enrolled 61 patients (28 COVID-19-positive and 33 controls) who were tested for other comorbidities and co-existence of associated oral pathogenic microbiota. Results: A total of 247 bacterial isolates were identified in the bacterial cultures in both groups. Viral hepatitis type A was more prevalent in the COVID-19-positive group (p = 0.026), as was the presence of oral candidiasis (p = 0.006). In the control group, a moderate direct relationship was observed between the Beta hemolytic streptococcus group G and dermatitis, and strong direct relationships were observed between the Beta hemolytic streptococcus group G and external otitis, Streptococcus pyogenes and dental alveolitis, and Streptococcus pyogenes and chronic lymphocytic leukemia. In the test group, strong direct relationships were observed between Hemophilus influenzae and pulmonary thromboembolism; Staphylococcus aureus and autoimmune thyroiditis; post-viral immunosuppression, chronic coronary syndrome, and hypernatremia; Beta hemolytic streptococcus group C and rheumatoid polyneuropathy; Beta hemolytic streptococcus group G and hyperkalemia, hypothyroidism, secondary anemia, and splenomegaly; and active oral candidiasis and SARS-CoV-2 viral pneumonia. The following relationships were strong, but inverse: Beta hemolytic streptococcus group G and acute respiratory failure, and active oral candidiasis and SARS-CoV-2 viral bronchopneumonia. Conclusions: Briefly, COVID-19-positive patients have the predisposition to build up associated comorbidities and coinfections, which can be the expression of the immune burden that this virus generates to the host.

The human coronavirus SARS-CoV-2, causing COVID-19 disease, is a respiratory virus that uses the oropharynx as the primary site of replication [3].The oral cavity, being the entry point to the body, may be an active site of infection and an important reservoir of SARS-CoV-2, playing a critical role in its pathogenesis; however, it is unclear whether SARS-CoV-2 infection can alter the local homeostasis of the resident microbiota, actively cause dysbiosis, or influence cross-body site interactions [4,5].Considering that the oral surfaces are colonized by a diverse microbial community (bacteria, viruses, and fungi), it is likely that viruses have interactions with the host microbiota and can also influence SARS-CoV-2 infection [4].Periodontal-associated cytokines might drive the alteration of the respiratory epithelium via the aspiration of oral pathogens into respiratory organs to promote the adhesion of the virus; therefore, the oral microbiome might impact lung infection and microbial colonization by SARS-CoV-2 [4,[6][7][8].
Bacterial coinfections are not uncommon with respiratory viral pathogens, and although they are rare upon patient admission, they are frequent among patients requiring prolonged hospitalization for COVID-19, particularly those who require mechanical ventilation in an ICU and can add to significant mortality and morbidity [9].
Although the disease results in mild symptoms in most cases, it progresses to severe pneumonia and multi-organ failure, leading to mortality in some cases depending on patient age and the presence of comorbidities [10][11][12][13][14][15].Independent risk factors, such as elevated procalcitonin and C-reactive protein levels, lymphopenia, leukocytosis, and acute kidney injury requiring dialysis, were identified as down-graders in SARS-CoV-2 viral infection evolution.
Additionally, more recent studies have reported clinical orofacial manifestations in COVID-19-positive patients, including oral ulcerative lesions, vesiculobullous lesions, and acute sialadenitis [1].Due to resource constraints, overburdened healthcare systems, and current evidence suggesting lower rates of coinfections, it is plausible that patients infected with COVID-19 are simply not being evaluated for coinfections.Thus, the true coinfection rate may be higher than what is currently suggested by the available literature [16].
The aim of this study was to identify specific rhino-and oropharyngeal microbiological pathogens as well as associated comorbidities that favor SARS-CoV-2 infection and corelate them.

Study Population
Between October 2021 and November 2021, this descriptive and analytical trial enrolled 61 subjects (33 males and 28 females) who were outpatients at the Clinical Hospital of Infectious Diseases and Pneumophysiology "Dr.Victor Babes , Timis , oara" and at the ENT Department of the Faculty of Medicine of the "Victor Babeş" University of Medicine and Pharmacy in Timis , oara.The study protocol was approved by the Research Ethics Committee of the "Victor Babeş" University of Medicine and Pharmacy (approval no.77/16.11.2021).
The study was conducted over a period of one and a half months (1 October 2021-12 November 2021) in accordance with principles outlined in the Declaration of Helsinki on experimentation involving human subjects.All subjects were informed about the nature and purpose of the study, and each subject signed an informed consent document giving their permission for the sampling of biological material.The study population consisted of males and females (mean age, 62.39 ± 15.61 years; range, 20 to 90 years).

Microbiological Sampling
Hospitalized COVID-19-positive patients (n = 28) and matched healthy controls (n = 33) underwent rhino-pharyngeal, oropharyngeal for diagnosis of SARS-CoV-2, and oral swabs specimens touching tongue, palate, and cheeks, and then they were additionally collected for oral microbiota identification.The history of hepatitis A was collected upon admission in the hospital evaluating each patient's medical history.The clinical standard admission protocol in the hospital requires complete blood counts and antibiogram.
Identification of SARS-CoV-2 was carried out using real-time polymerase chain reaction (real-time PCR) analysis performed on a BIO-RAD CFX96 Real-Time System C1000 Touch Thermal Cycler Device.For bacteria, isolation was conducted on Columbia 5% sheep blood agar (Sanimed, Bucharest, Romania).Identification of all isolates was performed according to the morphological characters of colonies and their biochemical tests obtained using the automated VITEK 2 system (bioMérieux, Inc., Marcy-l'Étoile, France).

Statistical Analysis
Minimum sample size was calculated using G*Power (v.3.1.9.6, Kiel University, Kiel, Germany) for the following parameters: power = 80%; effect size 0.5.The minimum calculated sample size was 52.The number of enrolled patients was larger than the minimum required sample size (n = 61).
Normality was assessed using the Shapiro-Wilk test, which was rejected for all parameters.As such, non-parametric tests were chosen.Results are presented as median values and interquartile ranges (IQRs).For comparison of these data, the Mann-Whitney U test was used.Associative testing was performed using Spearman's rank correlation, the interpretation of the results being observable in Table 1.Categorical data are presented as number and frequency.Contingency tables were created and the Chi 2 test was used to check for associations between table rows and columns.For cases of variable values below 5, Fisher's exact test was used instead.All p values < 0.05 were regarded as statistically significant.Statistical analysis was performed using MedCalc ® Statistical Software version 20.216 (MedCalc Software Ltd., Ostend, Belgium).
When checking for associations, using the Chi 2 test, the following relationships could be observed: COVID-19 and sex (p = 0.049), with more males being in the positive group; COVID-19 patients and history of viral hepatitis A (p = 0.03926), with history of viral hepatitis A being more prevalent in the positive group; and COVID-19 patients and active oral candidiasis (p = 0.0076), with more patients with active candidiasis being in the positive group.This is detailed in Table 2.

COVID-19-Negative Patients
Of the total 33 healthy controls, there were 14 (42.42%)males and 19 (67.86%) females.For this group, the Spearman's rank correlation test was used, and the statistically significant results are displayed in Table 3.As such, a moderate direct relationship was observed between Beta hemolytic streptococcus group G and dermatitis, and strong direct relationships were observed between Beta hemolytic streptococcus group G and external otitis, Streptococcus pyogenes and dental alveolitis, and Streptococcus pyogenes and chronic lymphocytic leukemia.

COVID-19-Positive Patients
Of the total 28 COVID-19-positive patients, there were 19 (67.86%) males and 9 (32.14%)females.For this group, the Spearman's rank correlation test was used, and the statistically significant results are displayed in Table 4. Moderate direct relationships were observed between Beta hemolytic streptococcus group C and hepatocytolysis and Beta hemolytic streptococcus group G and hyperkalemia, while a moderate inverse relationship was observed between Beta hemolytic streptococcus group G and asthenic syndrome.
Strong direct relationships were observed between Hemophilus influenzae and pulmonary thromboembolism, Staphylococcus aureus and the female sex, Staphylococcus aureus and autoimmune thyroiditis, Staphylococcus aureus and post-viral immunosuppression, Staphylococcus aureus and chronic coronary syndrome, Staphylococcus aureus and hypernatremia, Beta hemolytic streptococcus group C and rheumatoid polyneuropathy, Beta hemolytic streptococcus group G and hyperkalemia, Beta hemolytic streptococcus group G and hypothyroidism, Beta hemolytic streptococcus group G and secondary anemia, Beta hemolytic streptococcus group G and splenomegaly, and active oral candidiasis and SARS-CoV-2 viral pneumonia.The following relationships were strong, but inverse: Beta hemolytic streptococcus group G and acute respiratory failure, and active oral candidiasis and SARS-CoV-2 viral bronchopneumonia.

Discussion
Several recent studies highlighted the potential role of the oral microbiome in triggering different bacteria of viral infection; however, the particularity of this mechanism is vaguely defined.The novel concept of "the keystone pathogen" is considered to be involved in the complex process of dysbiotic diseases by coordinating the pathogenic microbiota trough the saprophytic one [17].
Early studies suggested that men and women were equally susceptible to COVID-19 infection; however, men are at higher risk for severe symptoms and death, a fact that is also observed in our research [18].This may be caused by the inherited differences in the systemic immune responses of the innate and adaptive immune system, rendering them more susceptible to an unfavorable response to infection [18][19][20].
In the present study, positive correlations were found between acute COVID-19 infection and hepatitis A virus presence, a parallel also demonstrated by other authors [21] and also with hepatitis B [22] and E [23].
The presence of Candida albicans in the oral cavity can be totally benign, being part of the usual commensal flora; however, its pathogenicity is validated as opportunistic when immune status is compromised, like in the case of COVID-19 infection [24].The incidence of oral candidiasis is frequent in hospitalized patients with COVID-19 that undergo broad-spectrum antibiotic or prolonged corticosteroid treatment [25][26][27][28].Our results also confirmed that there is a solid incidence of oral candidiasis in the positive group (p = 0.006).
Infection with Candida albicans in COVID-19 patients was also strongly correlated positively with pneumonia (R = 0.561, p = 0.002) but inversely with bronchopneumonia (R = −0.535,p = 0.003).This is important, as other studies have pointed out, to the risk that these patients have in regard to possible system infections due to fungal agents [29].Also, the relationship between SARS-CoV-2 viral pneumonia and oral candidiasis was observed by other authors as well [24].
Although, in our study, the Chi 2 test could not establish a relationship between bacterial agents and COVID-19 infection, which was observed by other authors [30][31][32][33], several relationships could be observed between the studied pathogens and several comorbidities, especially in the COVID-19-positive group.
Gram-positive pathogens like Streptococcus can induce various infections in humans.There are many different types of streptococci, their classification being made into groups based on their cell wall antigens.Infections with group C and G streptococci are far less common and understood than those with group A and B, yet they can cause pharyngitis [34], skin infections [35][36][37], and bacteremia [38].Even if there are no specific data supporting that streptococcus beta hemolytic group G clearly causes external otitis, our findings showed a strong relationship between these two (p < 0.001).
Another member of the Streptococcus family investigated in our research is Streptococcus pyogenes, of which its presence was strongly correlated with dental alveolitis, a fact confirmed by many other authors [39][40][41].Nevertheless, in immunosuppressant diseases like chronic lymphocytic leukemia, patients are prone to infections because of both humoral immunodepression, innate to the hematologic disease, and the immunosuppression related to the administrated treatment [42]; hence, the activity of co-infections is exacerbated.
Although prior studies identified several pathways contributing to pulmonary thromboembolism, it is unknown whether this is COVID-19-specific or also occurs in acute respiratory distress syndrome patients with another infection like Hemophilus influenzae [43].Nevertheless, COVID-19 patients are at an increased risk of pulmonary thromboembolism development [44].
COVID-19-positive women are more likely to develop Staphylococcus infections, as proved by a moderate direct relationship (R = 0.503, p = 0.006) in the Spearman's test.However, there are no data regarding the prevalence of S. aureus in women specifically [45,46].A multicenter, retrospective cohort study showed that Staphylococcus aureus bacteremia causes significant morbidity and mortality in patients with pre-existing immunosuppression, an aspect confirmed by our study as well [47].Staphylococcus aureus is not typically associated with chronic coronary syndrome, which is a pathological process characterized by atherosclerotic plaque accumulation in the epicardial arteries; however, some studies have shown that there may be an association between this latter cardiac pathology and bacterial infections such as Chlamydia pneumoniae and Helicobacter pylori [48][49][50].We found a strong correlation between chronic coronary syndrome and the presence of Staphylococcus aureus in the COVID-19-positive group.
Hypernatremia is a condition that generates an excess of sodium in the bloodstream and can be caused by many factors including dehydration, kidney disease, and certain medications like diuretics [51] and corticosteroids [52].Even though there is not any direct evidence in the literature that there is a link between Staphylococcus aureus and hypernatremia, it is possible that the infection with this pathogen could lead to dehydration, which might then lead to hypernatremia.
No direct link in the literature was found for Beta hemolytic streptococcus group C and hepatocytolysis; however, it is possible that this pathogen can cause liver damage in some cases [53].Interestingly, although a very important complication in COVID-19positive patients is represented by acute respiratory failure, it was inversely and strongly correlated (R = −0.694,p < 0.001) with the oral presence of Beta hemolytic streptococcus group G, which can cause respiratory infections in rare cases.The same can be said about the relationship between the presence of Beta hemolytic streptococcus group G in oral samples and the presence of asthenic syndrome; however, this relationship was considered to be moderate (R = −0.403.p = 0.034).No such relationships have been previously described; however, a shift in microbiota due to the severity of COVID-19 has been observed in the literature and may be the mechanism at play in this regard [54,55].Also, a study by Islam et al. pointed to a correlation of high association of streptococcus spp.with secondary bacterial lung infection, breathing difficulty, and sore throat [56].

Limitations
Firstly, although the minimum sample size was achieved and overcome, compared to other research, our sample size was moderate.A larger sample size would provide more in-depth information.Secondly, although we used the Vitek 2 automated system for the identification of microbial agents, identification using PCR or another molecular biology method might have provided more sensitive in this regard.

Conclusions
We can conclude that COVID-19-positive patients have a tendency to develop more associated comorbidities and coinfections.This might be the expression of the immune burden that this virus provokes to the host, which can be amplified by the effects of the medication for the treatment of this systemic viral infection.

Table 1 .
Interpretation of the R values.

Table 2 .
The results of the contingency table testing.

Table 3 .
The results of the Spearman's rank correlation test for healthy controls.

Table 4 .
The results of the Spearman's rank correlation test for the positive group.