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ANTIBIOTIC RESISTANCE IN COMMUNITY-ACQUIRED PNEUMONIA. A ROMANIAN PERSPECTIVE

Community-acquired pneumonia (CAP) is one of the most common communicable diseases with a high mortality rate worldwide. Treatment is administered empirically based on clinical symptoms and the most commonly incriminated microorganisms in lower respiratory infections responsible for CAP. This study aimed to identify microorganisms responsible for CAP in patients hospitalized in an Emergency Hospital in Bucharest, Romania; to assess the impact of patients’ comorbidities and age on the survival level and to establish the degree of antimicrobial resistance of the most important isolated species. A total of 170 patients diagnosed with CAP from December 2017 through December 2018 were studied. Statistical analyses were performed considering the following data: survival rate, comorbidities, initiated pharmacotherapy, antibiogram results in case of refractory treatments. The overall survival rate was 36% among patients. Cardiovascular disease was incriminated as the highest risk factor. Concerning refractory antibiotic treatment, the microbiological results revealed that the most common bacterial strains detected were Klebsiella sp., Acinetobacter baumanii, S. aureus, E. coli and Pseudomonas, with high multiple antibiotic resistance (MAR) index. The results point out a real concern for patients with refractory CAP due to high antimicrobial resistance to the administered antibiotics in Romania.


Introduction
Community-acquired pneumonia (CAP) is an acute infection of the lower respiratory tract with a high mortality rate worldwide. The diagnosis of community-acquired pneumonia can be established in patients without any contact with hospitals or other health care settings over a 3 months period before hospitalization [1,2]. CAP is one of the most common causes of hospitalization for an infectious disease worldwide. A retrospective study from the Netherlands undertaken between 2008 and 2011 pointed out an incidence of 295 cases of CAP per 100,000 persons per year [3]. In USA, approximately 650 patients with CAP per 100,000 population are diagnosed yearly and 9% of these patients are hospitalized at least once more during the same year due to a new episode of CAP [4,5]. The main risk factors for CAP are older age, viral respiratory infections, smoking, excessive alcohol consumption, chronic comorbidities (such as chronic obstructive pulmonary disease, asthma, bronchiectasis, congestive heart failure, diabetes mellitus, immunosuppressive diseases, stroke), or factors related to the lifestyle (living in shelters, institutionalized elderly patients, exposure to toxins) [6][7][8].
The aetiology of CAP includes: typical microorganisms, atypical microorganisms, and respiratory viruses. The typical microorganisms involved are Staphylococcus aureus, aerobic Gram-negative bacteria, group A streptococci, or microaerophilic bacteria. Other microorganisms, such as Legionella spp., Chlamydia pneumoniae, Mycoplasma pneumoniae, Chlamydia psittaci, Coxiella burnetti, are those resistant to betalactams or those that cannot be cultured by traditional methods or cannot be visualized in Gram stain. Apart from influenza and rhinovirus, the viral agents involved in the pathophysiology of CAP may be parainfluenza viruses, adenoviruses, coronaviruses and respiratory syncytial viruses. The prevalence of these microorganisms depends on the geographical area, the season, the vaccination rates and the host comorbidities [9][10][11][12][13]. Despite the increasing use of microbiological tests and molecular diagnostic tests, in half of the cases of CAP, the pathological agent cannot be identified, meaning that treatment may be empirical [5,14]. CAP complications occur especially in patients with various comorbidities, the most common being respiratory failure, bacteraemia with antibiotic resistant germs or fungus from Aspergillum or Fusarium genus, parapneumonic pleurisy and rarely, pulmonary abscess [15 -18]. Selection of empiric antibiotherapy should be based on the proper antimicrobial resistance pattern knowledge in a particular geographical area, correlated with specific individual risk factors. Thus, the treatment targets the most probable microorganisms involved, such as: Streptococcus pneumoniae, Haemophylus influenzae, Chlamydia pneumoniae, Mycoplasma pneumoniae, Legionella sp., etc. [1,12,19,20]. In this context, the objectives of the present study are: i) to identify the microorganisms that produced CAP in patients hospitalized in an Emergency Hospital from Bucharest, Romania; ii) to establish the degree of antimicrobial resistance of the most important isolated species and iii) to assess the impact of patients' comorbidities and age on the survival level.

Subjects
The study was conducted between December 2017 and December 2018 and included a total of 170 patients consecutively hospitalized with CAP, during this time, at the Department of Internal Medicine within the Clinical Emergency Hospital, Bucharest, Romania. The inclusion criteria considered the patients diagnosed with CAP without any contact with hospitals or other health care settings over a 3 months period before hospitalization [1,2]. The exclusion criteria considered the patients which had been in contact with health care units or hospitals, three months prior diagnosis. Each patient included in the study provided written informed consent. The study was conducted in accordance with the World Medical Association Declaration of Helsinki and was approved by the Institutional Ethics Committee of Clinical Emergency Hospital of Bucharest. Sputum samples were collected and the microbiological analyses were performed by classical bacteriological diagnosis. The antimicrobial testing was performed by disc diffusion according to the guidelines of the Clinical and Laboratory Standards Institute [21]. Statistical analysis Biostatistical analysis was implemented using the R statistical software (R version 3.5.3). For a relevant statistical analysis, antibiotic resistance was calculated as the ratio between the number of isolates identified as resistant over the total number of isolates tested for a particular antibiotic [15,22]. Antibiotic resistance was also associated with multiple antibiotic resistance index (MAR) as a continuous type variable, computed for each isolate tested. MAR was calculated as a ratio between the number of antibiotics at which the isolate was resistant over the total number of tested antibiotics [15,22]. The continuous variables were compared using Student's t-test for two groups. For more than two analysed groups we use one-way or two-ways analysis of variance (ANOVA) depending on the situation, followed by multiple comparisons, if the p-value indicated statistically significant differences (a = 0.05). Categorical variables had been analysed by Pearsontype tests.

Results and Discussion
Data were collected from 170 patients (male to female ratio 1:1) and the survival rate was 36% among patients. The first part of the research focused on individuals suffering from pneumonia and different comorbidities impact in relation to survival level. Nine comorbidities were identified among patients ( Figure 1). Comorbidities with a particularly serious individual survival impact (Pearson's Chi-squared test with Yates' continuity correction) were cardiac (P < 0.001), pulmonary (P < 0.001), respiratory (P < 0.01) and diabetes (P < 0.05) (Figures 1 and 2). Flu and renal diseases did not influence the survival rate of the patients (P > 0.05).

Figure 10.
Resistance/sensitivity antibiotic profile determined for Staphylococcus aureus E. coli sensitivity profile ( Figure 11) revealed a high level of resistance to Ampicillin and Amoxicillin (80%) and antimicrobial activity for Amikacin, Cefoperazone, Meropenem, Piperacillin and Tigecycline.
In the case of Pseudomonas strains, the analyses ( Figure 12) revealed high-level resistance to ciprofloxacin and ceftazidime (75%), a decreased resistance to meropenem (20%) and sensitivity to piperacillin and colistin.  The clustering analysis showed 4 clusters for Acinetobacter baumannii; two in the sensitive area, probably with slightly decreased resistance and 2 in the high resistance area. (Figure 13a). Klebsiella analysis highlighted 3 clusters, one in the sensitive area and 2 in the high resistance area. (Figure 13b). Community acquired pneumonia is an infectious disease with a high mortality rate worldwide. Patient survival is affected by several factors like age and associated comorbidities. The aetiology of CAP includes most often bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, or viral pathogens, such as influenza or rhinovirus. In a large proportion of cases, the pathologic agent remains unidentified, despite a complete microbiological workup [23,24]. During the last years, changes in the prevalence of different etiological agents of CAP were observed. Due to the widespread use of pneumococcal vaccination, a decline of CAP prevalence with S. pneumoniae has been observed [10,25]. The prevalence of CAP with S. pneumoniae is higher in Europe than in the USA (30% versus 10 -15%), where the vaccination rates are also higher [25]. Nowadays, respiratory viruses are highly evoked as causes of CAP, based on the present available molecular diagnostic methods [14]. Due to the difficulties in the identification of the involved pathogenic microorganisms, guidelines recommend empirical antibiotic treatments, targeting the most probable bacterial strains [5,26]. The patients' survival rates in the current study were decreased mainly in the case of those aging between 60 -80 years old. Comorbidities encountered among patients and cardiovascular diseases were incriminated as the highest risk factor for CAP patients' outcome. Microbiological analyses were required in case of refractory antibiotic treatments. The results revealed that the most common bacterial strains detected were Klebsiella sp., Acinetobacter baumanii, S. aureus, E. coli and Pseudomonas. These types of pathogens are more commonly encountered in nosocomial infections and quite rare in CAP, according to the most recent clinical studies published. [1,5,27].
Among all European countries, Greece also faces one of the highest rates of carbapenem-resistant in Gram-negative bacteria [28]. In particular, from January 2011 through June 2012 a total of 7477 cases of carbapenem-resistant infections were notified in the frame of an Action Plan to combat infections due to carbapenem-resistant, Gram-negative pathogens, which translates to a mean incidence of 0.48 per 1,000 patient-days, and an associated crude 28-day mortality of 34.4% was recorded [28]. In response to these alarming indexes, the second phase of the Action Plan was implemented in all hospitals across the country, consisting of enhanced standard infection control practices, isolation of carriers and infected patients from non-carriers, and strict implementation of contact precautions [28]. Another concern about the bacteria resistance pattern resides in the geographical factors in the actual context of migration and different health systems intersections [29]. The antibiogram of the bacterial strains responsible for the treatment resistant CAP cases exhibit a concerning resistance profile to the more commonly recommended and prescribed antibiotics like Amoxicillin, Ampicillin and Ciprofloxacin [1,25,27,30]. The results of the present study showed that the most prescribed antibiotics were cephalosporines, penicillines and fluoroquinolenes but resistant strains were identified for some representatives of these classes. The bacteria from the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) were identified in CAP patients. This group of bacterial strains are not specific for this disease [7,31,32]. The highest antibiotic resistance according to MAR (multiple antibiotic resistant index) was detected for Acinetobacter baumanii, Klebsiella sp. and S. aureus, which are part of the ESKAPE group, namely the bacterial strains with known resistance to the present antibiotic treatment schemes. Several strains showed resistance to Meropenem and Amikacin, which were considered a solution to the refractory treated patients, raising real future concerns for the CAP diagnosed patients.

Conclusions
The present study pointed out a high mortality rate among patients diagnosed with community acquired pneumonia in Bucharest, Romania. The low survival rate depended on the age, associated comorbidities and the microorganisms responsible for the respiratory infection. In case of the refractory CAP, the microbiological analyses were performed and the results revealed the presence of some strains, specific for the nosocomial infections (Acinetobacter baumanii, Klebsiella sp., Escherichia coli, S. aureus and Pseudomonas sp.) with high resistance to the most commonly prescribed antibiotics.
To understand the pathogenic and clinical features of CAP, especially in the case of difficult to eradicate germs with high bacterial resistance, long-term clinical, genetic and epidemiological studies are required. It is imperative to establish a nation-wide surveillance system for resistant and multi-drug resistant pathogens in healthcare facilities and the community, in order to guide interventions. Patterns of antibiotic prescription in ambulatory services and hospitals should also be studied. The results of this study may be useful in drawing up "bacterial resistance maps".