Antibiotic resistance and biofilm formation in Klebsiella spp. isolates from Intensive Care Units in the Brazilian Amazon

Resistência aos antibióticos e formação de biofilme em isolados de Klebsiella spp. de Unidades de Terapia Intensiva na Amazônia Brasileira

Abstract

Klebsiella spp. is an opportunistic pathogen which poses a significant threat to public health, especially due to antimicrobial resistance and biofilm formation. This study aimed to determine the antibiotic resistance profile, biofilm formation and β-lactamases production in Klebsiella spp. strains from clinical samples obtained from hospitalized patients, health professionals and hospital environment of intensive care units (ICUs) in Brazilian Amazon. The strains were obtained from clinical samples in different hospitals and identified using molecular techniques. The antimicrobial susceptibility was investigated via disk diffusion and microdilution. Biofilm formation was evaluated using a microtiter plate assay, while the extended-spectrum β-lactamases (ESBL) and carbapenemases production was assessed via disk approximation tests and combined disk tests, respectively. A total of 226 Klebsiella spp. strains were identified, with 141 coming from patients hospitalized in ICUs, 54 from healthcare workers, and 31 from hospital structures. Collection sites that showed the highest frequencies of isolated bacteria were the armpit (43,3%), oral cavity (42.6%), nasal cavity (70.4%), beds (54.8%) and mechanical ventilation (19.4%). Klebsiella spp. isolates from hospitalized patients and hospital ICU environments showed a high frequency of resistance (>50%) to the antibiotics, cefuroxime, cefotaxime, ceftriaxone, ciprofloxacin and aztreonam, and greater sensitivity (>70%) to carbapenems, amikacin and polymyxin B. Samples obtained from hospital structures (74.2%) and patients (51.8%) exhibited a high rate of multidrug resistant (MDR) isolates. In addition, 29% of Klebsiella isolates were found to produce ESBL and 15.5% carbapenemases. Biofilm formation was observed in 58.4% (132/226) of the isolates, with percentages of 64.5% (91/141) in hospitalized patients, 51.6% (16/31) on hospital structures, and 46.3% (25/54) among healthcare professionals. These results indicated a high percentage of antibiotics resistance and MDR in isolates from hospital structures and patients, which also showed ability to produce biofilms, ESBL and carbapenemases. Our findings reinforce the need to monitor resistance and adopt measures aimed at preventing the spread of MDR bacteria in ICUs.

Keywords:
drug resistance; biofilm; Klebsiella; Intensive Care Units

Resumo

Klebsiella spp. é um patógeno oportunista que representa uma ameaça significativa para a saúde pública, especialmente devido à resistência antimicrobiana e formação de biofilme. Este estudo objetivou determinar o perfil de resistência aos antibióticos, formação de biofilme e a produção de β-lactamases em isolados de Klebsiella spp. provenientes de amostras clínicas obtidas de pacientes hospitalizados, profissionais de saúde e ambiente hospitalar de Unidades de Terapia Intensiva (UTIs) na Amazônia brasileira. Os isolados foram obtidos a partir de amostras clínicas em diferentes hospitais e identificados utilizando técnicas moleculares. A suscetibilidade antimicrobiana foi investigada através de disco-difusão e microdiluição. A formação de biofilme foi avaliada através de um ensaio em placa de microtitulação, enquanto a produção de β-lactamases de espectro estendido (ESBL) e carbapenemases foram analisadas através do teste de aproximação de disco e teste do disco combinado, respectivamente. Um total de 226 isolados de Klebsiella spp. foram identificados, sendo 141 provenientes de pacientes hospitalizados em UTIs, 54 de profissionais de saúde e 31 de estruturas hospitalares. Os locais de coleta que apresentaram maior frequência de bactérias isoladas foram a axila (43,3%), cavidade oral (42,6%), cavidade nasal (70,4%), leitos (54,8%) e aparelhos de ventilação mecânica (19,4%). Isolados de Klebsiella spp. de pacientes hospitalizados e ambiente hospitalar de UTIs mostraram uma alta frequência de resistência (>50%) aos antibióticos cefuroxima, cefotaxima, ceftriaxona, ciprofloxacina e aztreonam, e maior sensibilidade (>70%) aos carbapenêmicos, amicacina e polimixina B. Amostras obtidas de estruturas hospitalares (74,2%) e pacientes (51,8%) apresentaram alta taxa de isolados multirresistentes (MDR). Além disso, observou-se que 29% dos isolados de Klebsiella produziam ESBL e 15,5% carbapenemases. A formação de biofilme foi observada em 58,4% (132/226) dos isolados, com percentuais de 64,5% (91/141) em pacientes hospitalizados, 51,6% (16/31) em estruturas hospitalares e 46,3% (25/54) entre profissionais de saúde. Estes resultados indicam um elevado percentual de resistência aos antibióticos e MDR em isolados de estruturas hospitalares e pacientes, que também possuem a capacidade de produzir biofilmes, ESBL e carbapenemases. Nossos resultados reforçam a necessidade de monitoramento da resistência e adoção de medidas que visem prevenir a disseminação de bactérias MDR nas UTIs.

Palavras-chave:
resistência microbiana a medicamentos; biofilme; Klebsiella; Unidades de Terapia Intensiva

1. Introduction

Healthcare-associated infections (HAIs) constitute the most frequent adverse events in the context of health service delivery. These infections, when caused by multidrug-resistant (MDR) pathogens, may seriously harm patients, with a significant impact on morbidity and mortality, and in a significant financial burden to the healthcare system. The risk of contracting an HAI in an intensive Care Unit (ICU) is significantly higher, with an estimated 15 out of every 100 hospitalized patients from low- to middle-income countries, predicted to acquire at least one HAI during hospitalization, and on average 1 in 10 will die (WHO, 2022).

In addition to the risks to which hospitalized patients in ICUs are exposed, antimicrobial resistance (AMR) has emerged as a serious global public health threat. It is estimated that in the absence of preventive action, AMR may lead to 10 million deaths worldwide by 2050 (O’Neill, 2016). Notably, mortality among patients infected with resistant bacteria is two to three times higher than that of those infected with susceptible bacteria (WHO, 2022). In 2019, an estimated 4.95 million deaths were associated with AMR globally, with Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa causing more than 25,000 deaths each (Murray et al., 2022).

Considering the alarming scenario associated with bacterial resistance, the World Health Organization (WHO) has updated the “List of priority pathogenic bacteria” and classified Enterobacterales (including Klebsiella) resistant to carbapenems and third-generation cephalosporins, as a bacterial pathogen critical priority of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance (WHO, 2024).

Klebsiella species are an opportunistic gram-negative bacteria known to cause HAIs. In humans, these bacteria can cause soft tissue, bloodstream, urinary tract and surgical wound infections, as well as pneumonia, meningitis and sepsis (Dong et al., 2022). Klebsiella spp. are great concern in hospital settings, due to the development of antibiotic resistance and the possibility of being transmitted via person-to-person contact between patients and contaminated healthcare workers or through contaminated environments. In addition, patients may also become infected through medical equipment, such as mechanical ventilation devices or intravenous catheters, or through wounds caused by injuries or surgical procedures, which allow bacteria to enter the body and cause infections (CDC, 2023).

Klebsiella spp. exhibits intrinsic resistance to penicillin, producing different types of β-lactamase enzymes, such as SHV in K. pneumoniae. Moreover, this bacterial genus also has the capacity to acquire genetic elements through horizontal gene transfer (HGT) and mutations that confer antimicrobial resistance and/or virulence characteristics. The acquisition of multidrug resistance genes and the accumulation of resistance-associated mutations have made Klebsiella spp. a significant challenge for public health, particularly due to the increasing number of severe infections and the growing limitations in effective treatments (Dong et al., 2022).

β-lactams are considered the most widely used antimicrobial agents, being generally well tolerated and highly effective in eliminating resistant bacteria. (Vrancianu et al., 2020). However, due to the frequent use of these drugs, some gram-negative pathogens have developed resistance to β-lactams, especially via extended-spectrum β-lactamases (ESBLs) (Riwu et al., 2020). The ESBLs production constitute the predominant resistance mechanism in Klebsiella spp. as well as in other Enterobacterales, with TEM, SHV and CTX-M being the most prevalent (Bhaskar et al., 2019; Castanheira et al., 2021).

Carbapenems are often the last line of defense against infections by gram-negative pathogens that are resistant to other antibiotics. Nonetheless, the emergence and spread of bacteria resistant to these antimicrobials are associated with high mortality rates and potential to spread widely on account of resistance conferred by several types of carbapenemases, such as KPC, NDM, VIM and OXA-48 (Bhaskar et al., 2019; Elshamy and Aboshanab, 2020).

In addition to the production of β-lactamases, biofilm formation by Klebsiella species and several other gram-negative pathogens has been linked to antimicrobial resistance and persistence (Akinpelu et al., 2020). Biofilms are bacterial aggregates that are tightly bound and embedded in an extracellular matrix of polysaccharides, proteins, carbohydrates, enzymes and nucleic acid, that allows irreversibly anchoring to any surface (biotic and abiotic), being a natural resistance mechanism that enables these microorganisms to survive in certain environments (Guerra et al., 2022; Kuinkel et al., 2021). Infections or diseases caused by pathogens able to produce biofilms increase morbidity and mortality rates in patients (Assefa and Amare, 2022).

Porto Velho, the capital of the state of Rondônia, is located in the Western Amazon of Brazil and ranks last in the 2024 Basic Sanitation Ranking, where less than 50% of the population has access to treated water, and only 1.71% of sewage receives treatment (Trata Brasil, 2024). The lack of basic sanitation contributes to the spread of pathogens, increasing the incidence of infections, often caused by resistant microorganisms. In addition, hospitals in the region face issues such as a lack of human and material resources, absence of surveillance, and overcrowding, which are considered risk factors for the acquisition of HAIs. Hospital ICUs are viewed as environments conducive to the creation, dissemination, and amplification of antibiotic resistance (Maki and Zervos, 2021).

Thus, considering the pathogenic role of Klebsiella species in ICUs as well as the regional variability seen in the severity of AMR worldwide and the scarcity of associated data in the Brazilian Amazon, especially in Rondônia, we used samples obtained from hospitalized patients, health professionals and hospital environment of ICUs in Porto Velho, Rondônia, Brazilian Amazon, to determine the antibiotics resistance profile, biofilm formation and β-lactamases production in Klebsiella spp. strains.

2. Materials and Methods

2.1. Specimen collection, processing and bacteriology

During the periods from December 2017 to February 2018, December 2018 to January 2019, and November 2020, clinical samples were collected from hospitalized patients admitted to the ICUs, health professionals and hospital structures of the ICUs of three reference hospitals (named Hospital 1, 2 and 3) in Porto Velho City, Rondônia state, Brazil. This study was approved by the Ethics Committee of Tropical Medicine Research Center, Porto Velho, Rondônia, Brazil (Process n. 2.368.951).

Samples were collected from the oral cavity, tracheostomy secretions, armpit, wound secretions, blood and urine of hospitalized patients and from nasal mucosa secretion swabs and nails of health professionals. Swab samples were also collected from mechanical ventilation devices, beds, stretchers, operating room machines, computer keyboards, sinks, floors and water taps found in ICU environments.

All samples were transported to the Microbiology Laboratory of the Oswaldo Cruz Rondônia Foundation for processing and species identification. Swabs collected from hospital structures, health professionals and hospitalized patients (except blood and urine samples) were cultured in Luria-Bertani Broth (LB; Kasvi^®, Italy) for 18 to 24 h at 37±2° C in an orbital shaker at 100 rpm and subsequently seeded in blood agar (AS; HiMedia^®, India), McConkey (MC; Kasvi^®, Italy), chromogenic (CA; Kasvi^®, Italy) and Luria Bertania agar (LBA; Kasvi^®, Italy) and incubated at 37±2° C for 18 to 24 h. The blood and urine samples were directly seeded in culture media AS, MC, CA and LBA. All colonies suggestive of Klebsiella spp. were submitted for molecular identification.

2.2. Species identification

Molecular identification was performed by extracting genomic DNA via the phenol-chloroform method (Sambrook et al., 1989), followed by conventional PCR amplification using primers specific to 16S rRNA according Arruda et al. (2017). The negative control consisted of all PCR reagents except the DNA template and genomic DNA from E. coli ATCC^® 25922^TM was used as a positive control for all reactions. The amplicons were purified using a EasyPure® PCR Purification kit (Transgen Biotech^®), according to the manufacturer's specifications and quantified using a NanoDrop1000^® (Thermo Scientific^®). DNA sequencing was carried out using the ABI 3100 Prism automatic sequencer (Applied Biosystems, USA) using the Sanger method.

Consensus sequences were obtained and analyzed using BioEdit Sequence Alignment Editor software (version 7.0) and bacterial species were identified via alignment with the Basic Local Alignment Search Tool (BLAST) database.

2.3. Antimicrobial susceptibility testing

Testing for antimicrobial susceptibility via the disk diffusion method was conducted in Mueller-Hinton agar (Kasvi^®, Italy), according to Clinical and Laboratory Standards Institute (CLSI) stipulations for the following antibiotics (Oxoid^TM): gentamicin (GEN - 10μg); amikacin (AK - 30μg); piperacilllin/tazobactam (TZP – 100/10μg); amoxycillin/clavulanic acid (AMC, 20/10μg); meropenem (MEM - 10μg); imipenem (IPM - 10μg); ertapenem (ETP - 10μg); ceftazidime (CAZ - 30μg); cefotaxime (CTX - 30μg); cefepime (FEP- 30μg); ceftriaxone (CRO - 30μg); cefuroxime (CXM - 30μg); ciprofloxacin (CIP - 5μg); levofloxacin (LEV - 5μg); aztreonam (ATM - 30μg); and ampicillin/sulbactam (SAM - 10/10μg). E. coli ATCC^® 25922^TM was employed for quality control in all tests. For the purposes of this study, MDR was defined as non-susceptibility to at least one agent in three or more antimicrobial categories (Magiorakos et al., 2012).

The Policimbac microdilution method (Probac®, Brazil) was used according to the manufacturer’s guidelines to determine the minimum inhibitory concentration (MIC) of the antimicrobial polymyxin B. E. coli ATCC^® 25922^TM and P. aeruginosa ATCC^® 27853^TM were employed as quality control.

2.4. Extended-spectrum β-lactamases (ESBLs) detection

For phenotypic detection of ESBLs, the disk approximation tests were used according to the method described by Kaur et al. (2013) with the following antibiotics (Oxoid^TM): amoxycillin/clavulanic acid (AMC - 20/10μg), cefotaxime (CTX - 30μg), aztreonam (ATM - 30μg), ceftriaxone (CRO - 30μg) and ceftazidime (CAZ - 30μg). K. pneumoniae ATCC^® 700603^TM was used as a control positive strain while E. coli ATCC^® 25922^TM was used as a negative control.

2.5. Phenotypic detection of carbapenemases

To detect carbapenemases, the combined disc test was used with the antibiotics ETP (10μg), IMP (10μg) and MEM (10μg), with or without the addition of enzyme blockers according to the methodology described in Technical Note No. 01/2013 of the National Health Surveillance Agency (ANVISA), a regulatory agency in Brazil (Brasil, 2013). IMP and MEM discs were impregnated with phenylboronic acid (AFB) for Ambler class A, while ethylenediaminetetraacetic acid (EDTA) was used for class B and phenylboronic acid and cloxacillin (CLOXA) for class C.

E. coli ATCC^® 25922^TM and other strains obtained from the Collection of Hospital Bacteria Cultures - CCBH (http://ccbh.fiocruz.br/), such as K. pneumoniae CCBH16302 (bla_NDM) and K. pneumoniae CCBH 6556 (bla_KPC), were used as quality control.

2.6. Biofilm production assays

Biofilm formation was assessed using 96-well polystyrene microplates in triplicate for each isolate, as described by Alvim et al. (2019), with modifications. The LB and E. coli ATCC^® 25922^TM were used as the negative control while P. aeruginosa ATCC^® 27853^TM was used as the positive control. Klebsiella spp. and controls strains were grown overnight in LB (Kasvi^®, Italy) for a period of 18 to 24 h at 37±2° C, in an orbital shaker at a speed of 100 rpm, following which the cultures were diluted 1:20 in LB, and inoculated (200μl) into 96-well polystyrene microtiter plates (Costar, USA).

Plates were incubated at 37º C for 20 h without shaking. Following incubation, the LB and unattached bacteria were removed by washing twice with 200 μl of distilled water. Adherent bacteria were stained for 5 min with 100 μl of 0.1% (w/v) crystal violet. Subsequently, the wells were washed twice with distilled water, unbound crystal violet was removed while the bound amount was released from stained cells using 100 μl of 95% ethanol. Biofilm was quantified by measuring absorbance at 570 nm (optical density, OD) using a microplate reader (BioTek Epoch microplate spectrophotometer).

Results were interpreted by averaging the optical density values of the triplicates, the cut-off point being calculated by averaging the OD of the negative control wells plus three times the standard deviation of the negative control to differentiate the strong/moderate biofilm producers from weak/non-producers.

2.7. Statistical analysis

The non-parametric tests, Fisher's Exact Test and Odds Ratio, were used for statistical analyses using Graph Prism 9.0, due to the use of convenience sampling and because they did not meet the requirements for parametric tests, such as small sample size and absence of normal distribution. The data was organized in a contingency table and the statistical significance was set at p<0.05.

3. Results

Of a total of 1,511 (100%) bacterial isolates, 226 (15%) were identified as belonging to genus Klebsiella, with 141 (9.3%) coming from patients hospitalized in ICUs, 54 (3.5%) from healthcare workers, and 31 (2.1%) from hospital structures.

K. pneumoniae was the species most frequently identified in hospitalized patients, with 78% (110/141) of isolates, followed by K. quasipneumoniae with 10.6% (15/141), K. aerogenes with 6.4% (9/141), K. variicola with 4.3% (6/141) and Klebsiella spp. with 0.7% (1/141). The armpit and oral cavity were the collection sites with the highest frequency of bacterial isolation, with percentages of 43.3% (61/141) and 42.6% (60/141) respectively. Furthermore, 7.1% (10/141) had tracheostomy secretion, 3.5% (5/141) urine and 3.5% (5/141) blood as origins.

Among health professionals, 38.9% (21/54) of the isolates belonged to K. aerogenes, 29.6% (16/54) to K. pneumoniae, 9.3% (5/54) to K. quasipneumoniae, 9.3% (5/54) to K. oxytoca, 7.4% (4/54) to K. variicola, 3.7% (2/54) to Klebsiella sp. and 1.9% (1/54) to K. michiganensis, with the majority coming from the nasal cavity (70.4% - 38/54) and the rest (29.6% - 16/54) from the nails.

K. pneumoniae isolates was predominant in hospital structures with a frequency of 90.3% (28/31), followed by K. aerogenes with 6.5% (2/31), and K. quasipneumoniae with 3.2% (1/31). These isolates were obtained from samples originating from beds (54.8%), mechanical ventilation devices (19.4%), water taps (12.9%), sinks (9.7%) and floors (3.2%).

Antimicrobial susceptibility profiles indicated that isolates of Klebsiella spp. from hospitalized patients displayed a high frequency of resistance to the antibiotics cefuroxime (58.9%), cefotaxime (57.4%), ceftriaxone (56%), ciprofloxacin (56%) and aztreonam (50.4%), of which 51.8% (73/141) showed MDR. On the other hand, it was observed that most showed sensitivity to polymyxin B (94.3%), as well as the carbapenems, ertapenem, meropenem and imipenem, and amikacin, with percentages higher than 70% (see Figure 1).

Figure 1
Antibiotic resistance profile in Klebsiella spp. clinical isolates from ICUs. Subtitle: SAM: ampicillin/sulbactam; TZP: piperacilllin/tazobactam; AMC: amoxycillin/clavulanic acid; FEP: cefepime; CTX: cefotaxime; CAZ: ceftazidime: CRO: ceftriaxone; CXM: cefuroxime; ETP: ertapenem; IPM: imipenem; MEM: meropenem; ATM: aztreonam; CIP: ciprofloxacin; LEV: levofloxacin; AK: amikacin: GEN: gentamicina; POL B: polymyxin B.

With respect to samples obtained from the hospital environment of ICUs, the bacteria showed resistance to the same antibiotics, but a higher proportion of bacteria were resistant to ciprofloxacin (80.6%), cefuroxime (77.4%), cefotaxime (74.2%), ceftriaxone (74.2%) and aztreonam (61.3%), of which 74,2% (23/31) were MDR (see Figure 1). In addition, a similarly high rate of isolates sensitive to polymyxin B (93.5%), carbapenems (> 70%), and amikacin (67.7%) were detected.

In contrast, the quantity of resistant isolates in samples obtained from healthcare professionals was considerably lower than that observed in patients and hospital structures, except by the greater resistance shown to the antibiotic amoxycillin/clavulanic acid (40.7%) (as show in Figure 1). Furthermore, a high percentage of sensitive isolates, especially to carbapenems, with 100% sensitivity to ertapenem, 96.3% to imipenem and 92.6% to meropenem, followed by aminoglycosides as amikacin (96.3%) and gentamicin (94.4%), and polymyxin B (94.4%). MDR was evidenced in 13% (7/54) of isolates.

ESBL production was phenotypically detected in 30.5% (43/141) of Klebsiella spp. isolates from hospitalized patients, in 11.1% (6/54) in those from intensive care workers, and 51.6% (16/31) in those from ICU environments. This result indicated a statistically significant association between the production of this type of enzyme and resistance to ampicillin/sulbactam, cefuroxime, cefotaxime, ceftriaxone, cefepime, ceftazidime and aztreonam (p<0.0001). Furthermore, a statistically significant association was observed between ESBL production and multidrug resistance in bacterial isolates from hospitalized patients (p= 0.0001; OR= 4.785), healthcare professionals (p= 0.0015; OR= 30) and hospital structures (p= 0.0155; OR= 13.13).

All bacterial isolates were phenotypically tested for carbapenemases, resulting in Class A serinocarbapenemase production (possibly KPC enzyme) being detected in 26 (18.4%) isolates from hospitalized patients, 6 (19.4%) from hospital structures, and 3 (5.6%) from healthcare worker samples. Producers of class B and class C carbapenemases were not detected. In addition, phenotypic Class A serinocarbapenemase production was found to be significantly related to multidrug resistance (p<0.0001; OR=60.12).

Biofilm production was evidenced in 58.4% (132/226) Klebsiella spp. isolates that were classified as strong/moderate producers, of which 64.5% (91/141) were from hospitalized patients, 46.3% (25/54) from healthcare professionals, and 51.6% (16/31) from hospital structures. Armpit (31.9%), oral cavity (27%), nasal mucosa (33.3%) and beds (29%) were the sources with the highest percentages of biofilm-forming isolates. The OD values by clinical sample origin are shown (see Figure 2). Analyses did not indicate a statistical association between non-susceptibility to the tested antibiotics and the ability to form biofilms. Similarly, no association was found between biofilm production and multidrug resistance. The frequency of biofilm-forming bacterial isolates that were classified as MDR or non-MDR are shown (see Table 1).

Figure 2
Classification of biofilm formation in Klebsiella spp. isolates from ICUs for clinical sample origin. (A) Non-biofilm forming strains; (B) Biofilm-forming strains.

Table 1
Biofilm formation in MDR and non-MDR Klebsiella spp. clinical isolates from ICUs.

Clinical isolates	Biofilm model
Hospitalized patients	Strong/moderate producer	Weak/non-producers
MDR	39 (27.7%)	34 (24.1%)
Non-MDR	52 (36.9%)	16 (11.3%)
Health professionals
MDR	4 (7.4%)	3 (5.6%)
Non-MDR	21 (38.9%)	26 (48.1%)
Hospital structures
MDR	11 (35.5%)	12 (38.7%)
Non-MDR	5 (16.1%)	3 (9.7%)
Total
MDR	54 (23.9%)	49 (21.7%)
Non-MDR	78 (34.5%)	45 (19.9%)

Based on the overall results of this study, different patterns of non-susceptibility to antimicrobials were evaluated per type of hospital, clinical samples, bacterial species, ability to form biofilms, ESBL production and carbapenemases (see Table 2). For example, the Hospital 2 showed the highest percentages of non-susceptibility to antibiotics compared with Hospital 1 and Hospital 3, as well as a high number of Klebsiella spp. MDR isolates (54.9%).

Table 2
Profile of Klebsiella spp. isolates not susceptible to antibiotics according to hospital of origin, site of collection of clinical samples, bacterial species, biofilm formation capacity, ESBL and carbapenemase production from ICUs.

Antibiotic	AMC	TZP	SAM	CAZ	CTX	FEP	CRO	CXM	ATM	CIP	LEV	GEN	AK	ETP	MEM	IPM	POL B	MDR
	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)
Hospital
Hospital 1 (n= 81)	63	30.9	55.6	38.3	43.2	43.2	43.2	66.7	38.3	45.7	39.5	21	19.8	19.8	29.6	21	2.5	38.3
Hospital 2 (n= 69)	30.4	43.5	65.2	23.2	63.8	63.8	63.8	85.5	58.0	72.5	56.5	46.4	36.2	20.3	18.8	17.4	13	59.4
Hospital 3 (n= 76)	38.2	7.9	43.4	44.7	50.0	46.1	48.7	63.2	44.7	42.1	35.5	31.6	7.9	13.2	13.2	13.2	2.6	40.8
Clinical Sample Origin
Oral cavity (n= 60)	46.7	33.3	50	43.3	55	55	33.3	78.3	53.3	91.7	50	35	25	20	20	18.3	1.7	53.3
Armpit (n= 61)	52.5	29.5	55.7	47.5	55.7	54.1	57.4	78.7	45.9	96.7	39.3	29.5	19.7	18	23	14.8	11.5	39.3
Tracheostomy secretion (n= 10)	80	60	80	70	70	70	70	90	70	90	80	80	50	50	50	50	0	80
Urine (n= 5)	80	80	100	80	100	100	100	100	100	100	80	40	40	40	40	40	0	100
Blood (n= 5)	60	80	80	60	60	60	60	100	60	100	100	80	0	60	60	60	0	80
Nails (n=16)	25	18.8	37.5	31.3	37.5	37.5	37.5	50	31.3	100	0	18.8	0	0	31.3	12.5	0	31.3
Nasal mucosa (n= 38)	52.6	5.3	21.1	2.6	13.2	7.9	7.9	31.6	5.3	97.4	13.2	0	5.3	0	0	0	7.9	5.3
Bed (n=17)	64.7	35.3	94.1	76.5	76.5	76.5	76.5	94.1	76.5	94.1	82.4	58.8	29.4	17.6	17.6	17.6	5.9	70.6
Mechanical ventilation device (n=6)	66.7	50	100	66.7	83.3	83.3	83.3	83.3	66.7	100	50	50	50	50	50	50	0	83.3
Sink (n= 3)	100	33.3	33.3	33.3	66.7	66.7	66.7	66.7	66.7	100	33.3	33.3	0	0	0	0	33.3	66.7
Water tap (n= 4)	100	75	100	75	75	75	75	75	75	100	75	75	50	0	0	0	0	75
Floor (n= 1)	100	100	100	100	100	100	100	100	100	100	100	0	0	100	100	100	0	100
Species
Klebsiella aerogenes (n= 32)	75	0	25	0	3.1	0	3.1	43.8	0	100	3.1	0	0	0	3.1	0	6.3	0
Klebsiella pneumoniae (n=154)	35.7	37.7	63	57.8	65.6	63.6	64.9	81.2	58.4	95.5	55.8	40.9	25.3	23.4	24.7	21.4	7.1	57.8
Klebsiella quasipneumoniae (n=21)	57.1	42.9	57.1	57.1	52.4	57.1	52.4	76.2	57.1	90.5	47.6	42.9	28.6	14.3	0	14.3	0	47.6
Klebsiella michiganensis (n=1)	100	100	100	100	100	100	100	100	100	100	0	0	0	0	100	0	0	100
Klebsiella oxytoca (n= 5)	40	40	40	20	40	40	40	40	20	100	0	0	0	0	60	40	0	20
Klebsiella sp. (n=3)	100	33.3	100	33.3	33.3	33.3	33.3	66.7	33.3	100	33.3	33.3	66.7	33.3	33.3	33.3	0	66.7
Klebsiella variicola (n=10)	0	0	0	0	0	0	0	10	0	10	0	0	0	0	0	0	0	0
Biofilm
Strong/moderate producer (n= 132)	46.2	23.5	49.2	44.7	49.2	48.5	50	72.7	43.9	18.2	41.7	25	12.1	10.6	11.4	9.1	6.1	40.9
Weak/non-producers (n= 94)	64.9	42.6	61.7	47.9	55.3	53.2	53.2	69.1	50.0	51.1	45.7	42.6	33.0	27.7	34.0	28.7	5.3	52.1
ESBL
Producer (n= 65)	60	26.2	84.6	83.1	100	27.7	100	100	84.6	83.1	63.1	56.9	18.5	3.1	1.5	4.6	4.6	80
Non-producer (n= 161)	51.6	33.5	42.2	31.1	32.3	31.1	31.7	59.6	31.1	40.4	35.4	22.4	20.5	23.6	28.6	22.4	6.2	31.7
Carbapenemases
Producer (n= 35)	94.3	100	100	97.1	100	100	100	100	97.1	88.6	88.6	65.7	60	85.7	100	88.6	11.4	97.1
Non-producer (n= 191)	46.6	18.8	46.1	36.6	42.9	41.4	40.3	66.0	37.2	46.1	35.1	26.2	13.6	5.2	6.3	4.2	4.7	36.1

• Isolates classified as intermediate and resistant were considered not susceptible to antibiotics. AMC: amoxycillin/clavulanic acid; TZP: piperacilllin/tazobactam; SAM: ampicillin/sulbactam; CAZ: ceftazidime; CTX: cefotaxime; FEP: cefepime; CRO: ceftriaxone; CXM: cefuroxime; ATM: aztreonam; CIP: ciprofloxacin; LEV: levofloxacin; GEN: gentamicina; AK: amikacin; ETP: ertapenem; MEM: meropenem; IPM: imipenem; POL B: polymyxin B; MDR: multidrug-resistant;. Clinical samples origin of the hospitalized patients: oral cavity. armpit. tracheostomy secretion. wound secretion. urine and blood; health professionals: nasal mucosa and nails; Hospitals structures: bed. mechanical ventilation device. sink. floor and water tap.

With respect to clinical samples from hospitalized patients, it was observed that microorganisms obtained from blood, urine and tracheostomy secretions had the highest percentage of bacteria not susceptible to antibiotics, with the rate of multidrug resistance being greater than 80%. Similarly, those from all hospital structure collection sites showed high frequencies of non-susceptibility to antibiotics with a MDR frequency > 65%. Samples obtained from the nails of professionals generally showed higher frequencies of non-susceptibility, compared to those from nasal mucosa (as show in Table 2).

Furthermore, K. pneumoniae, which was the main pathogen isolated from ICUs, showed high percentages of non-susceptibility to most antibiotics tested, while K. aerogenes showed low frequencies of resistance, with no MDR strains (see Table 2). Thus, our data, indicated that isolates producing ESBL and carbapenemases were associated with high percentages of MDR, with values of 80% and 97.1%, respectively (as show in Table 2).

4. Discussion

The hospitals are characterized as a high-risk environment for the development of HAIs, especially in ICUs. Klebsiella spp. is one of the main pathogens found in the hospital environment and has the ability to acquire genetic elements and mutations that confer resistance to antibiotics, leading to the emergence of MDR strains that are involved in an increasing number of cases of serious infections (Dong et al., 2022).

In the present study, we observed that most identified isolates belonged to K. pneumoniae, mainly in patients (78%) and in hospital structures (90.3%). This result may be attributable to K. pneumoniae belonging to the ESKAPE group of pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.), which are frequently isolated from clinical environments, capable of causing infections and linked to increased levels of resistance to various classes of antibiotics. In addition, K. pneumoniae is bacterial pathogen that is most commonly associated with HAIs, especially in patients in ICUs, as reported in different studies (Mota et al., 2018; Flores et al., 2020; Bshabshe, 2020; Denissen et al., 2022).

The armpit and oral cavity of patients, the nasal cavity of healthcare professionals, and beds and mechanical ventilation devices of hospital structures represented the collection sites with the highest frequency of bacterial isolation in the current study. This result was substantiated by literature, which describes Klebsiella spp. as opportunistic pathogens that are commonly found in the flora of the nose, throat, skin, and intestinal tract of healthy individuals, thereby justifying a higher percentage of this organism being detected in the above-mentioned sites (Dong et al., 2022).

In relation to hospital structures, a study with samples collected from different environmental surfaces and hospital structures of ICUs in Iran conducted by Tajeddin et al. (2016) demonstrated that surfaces frequently contaminated by bacteria were mechanical ventilation devices (82.91%), oxygen masks of patients (81.81%) and bed linen (67.65%), and that K. pneumoniae was the pathogen that mainly colonized mechanical ventilation devices (54.4%). In addition, some authors have shown that surfaces in proximity to patients are easily contaminated and with risk for transferring these pathogens (Hassan et al., 2019; Cruz-López et al., 2020).

Regarding the antimicrobial susceptibility profile, isolates of Klebsiella spp. from hospitalized patients showed a high frequency of resistance (>50%) to the antibiotics, cefuroxime, cefotaxime, ceftriaxone, ciprofloxacin and aztreonam, and greater sensitivity to carbapenems, amikacin and polymyxin B. Similar percentages of resistance to cephalosporins and monobactams with frequencies between 60-65%. were observed in a study conducted by Vasaikar et al. (2017). In addition, the authors observed low rates of resistance to carbapenems and amikacin.

Thuy et al. (2018) conducted a study of patients hospitalized in the ICU of a tropical disease hospital in Vietnam, and found relatively lower frequencies of antibiotic resistance in Klebsiella spp., compared to the present studies involving samples from Porto Velho, as indicated by percentages < 30% for ceftazidime, ceftriaxone, cefepime, ciprofloxacin and amoxycillin/clavulanic acid and a 3.6% percentage in isolates resistant to carbapenems. Other studies conducted in Brazil with Klebsiella isolates from patients hospitalized in the ICU showed a high prevalence of resistance to cephalosporins (80 - 100%), fluroquinolones (>60%) and carbapenems (>40%); (Ferreira et al., 2019; Gonçalves et al., 2017). These data demonstrated that the antibiotics susceptibility profile was variable between countries and regions, reinforcing the need for constant AMR monitoring.

Samples obtained from hospital structures contained a higher number of resistant isolates compared to those obtained from patients, with percentages of 80.6%, 77.4%, 74.2%, 74.2% and 61.3% for cefuroxime, cefotaxime, ceftriaxone and aztreonam, respectively. The presence of resistant microorganisms in ICU structures is worrying because contaminated surfaces and equipment have been identified as sources of transmission and dissemination of these pathogens. Therefore, we reinforce the need for proper sanitization/sterilization of surfaces, equipment and medical devices in the hospital environment to control these pathogens (Martin and Bachman, 2018).

In contrast to what was observed in patients and hospital structures, a low frequency of resistant bacteria and consequently of MDR was observed among healthcare workers samples (13%). This result is important because healthcare workers may serve as a reservoir for the transmission of bacteria, including resistant strains, in hospital environments, placing hospitalized patients at increased risk (Martin and Bachman, 2018; Elkady et al., 2022;).

In the present study, ESBL production was detected phenotypically in Klebsiella spp. isolates from hospitalized patients (30.5%), intensive care workers (11.1%) and hospital structures (51.6%). Other authors have also reported the production of ESBL enzymes with variable percentages (Frediani et al., 2023; Mulki et al., 2017; Tadesse et al., 2022; Vasaikar et al., 2017). Thuy et al. (2018) detected ESBL in 13.7% of Klebsiella spp. isolates in Vietnam. Bhaskar et al. (2019) demonstrated that 84% of K. pneumoniae samples from ICUs of a tertiary hospital in South India, were ESBL producers.

Moreover, a high rate of MDR isolates was evidenced in samples from hospital structures (74.2%) and patients (51.8%), with a statistically significant association being found between ESBL production and MDR. These data emphasize the need for continuous monitoring of the production of this enzyme in all hospitals and the rational use of antimicrobials. Notably, some studies have reported that hospitalized patients in the northern region of Brazil carried high frequencies of MDR K. pneumoniae, as seen in states such as Tocantins (84%) and Amazonas (61.9%; with 23.8% classified as extensively drug-resistant - XDR) (Ferreira et al., 2019; Nakamura-Silva et al., 2022).

In the present study, we evidenced the phenotypic production of Class A serinocarbapenemases (possibly KPC-type) in isolates from hospitalized patients, hospital structures, and healthcare workers, this phenotype being statistically associated with MDR. KPCs are the most common carbapenemases in cases of HAIs, and the detection of other serinocarbapenemases is rare (Elshamy and Aboshanab, 2020). The first KPC enzyme was obtained from a K. pneumoniae clinical isolate and characterized in North Carolina, USA, in 1996 (Yigit et al., 2001), following of various reports this enzyme which variable frequencies (Biberg et al., 2015; Viegas and Soares, 2018; Bhaskar et al., 2019; Ferreira et al., 2019). Furthermore, KPC-producing bacteria are usually resistant to other antimicrobials, such as aminoglycosides, fluoroquinolones and trimethoprim sulfamethoxazole, confirming their MDR status (Elshamy and Aboshanab, 2020).

In the present study, biofilm production was evidenced in 64.5% of Klebsiella spp. isolates from hospitalized patients, 46.3% from healthcare professionals and 51.6% from hospital structures samples. Yazgan et al. (2018) reported that 100% of the K. pneumoniae isolated from ICU patients in a Hospital in Turkey were biofilm formers. In Iran, Shadkam et al. (2021) evidenced that 75% of the K. pneumoniae strains were biofilm producers, with 25% and 19% being strong and moderate producers, respectively. Karimi et al. (2021) found that 74.5% of isolates from Hamadan hospitals had the ability to form biofilm, 21.6% moderately and 20.4% strongly. In Indonesia, Nirwati et al. (2019) observed that 85.63% of the K. pneumoniae isolates were producing biofilm, 26.95% strongly and 28.74% moderately.

The ability of bacteria to form biofilms in hospitals is problematic due to the risk of infections in hospitalized patients and because it contributes to the emergence of MDR microorganisms, which occurs due to the restricted penetration of antibiotics into the biofilm matrix, the presence of persistent cells, overexpression and exchange of resistance genes between bacteria, among other factors (Assefa and Amare, 2022). In hospitals, biofilms can be formed in the hospital wastewater, solid surfaces, drugs and medical instruments, leading to HAIs, with K. pneumoniae being one of the most prevalent biofilm-forming bacteria. It is estimated that biofilms are responsible for more than 65% of nosocomial infections, approximately 80% of chronic infections, and 60% of all human bacterial infections (Assefa and Amare, 2022; Chowdhury et al., 2018).

In contrast to previous studies (Karimi et al., 2021; Shadkam et al., 2021), the present study found no statistical association between non-susceptibility to antibiotics and/or MDR, and the ability to form biofilms. Our results are substantiated by Nirwati et al. (2019) who did not find a significant association between MDR K. pneumoniae and biofilm production capacity. It has been suggested that bacterial isolates susceptible to antibiotics may have a tendency to form stronger biofilms compared to those that are resistant, biofilm formation being an important factor for the survival of susceptible strains (Shadkam et al., 2021).

Although this study presented relevant results on the phenotypic detection of ESBL and carbapenemases, and is a widely accepted approach in clinical practice for the management of antimicrobial therapy, subsequent molecular studies are needed to identify resistance genes (EUCAST, 2017). The detection of resistance genes is extremely important, considering that during the pandemic antibiotics were prescribed excessively without necessity and that in 2022 Anvisa published technical note no. 74, which reports an increase in the frequency of isolation of MDR bacteria and simultaneous detection of the bla_KPC and bla_NDM genes, especially in the Klebsiella pneumoniae complex (59.5%). In addition, it was observed that during the pandemic period the number of isolates with this profile increased nearly six times. These data are alarming and present a new challenge to be confronted: the coexpression of carbapenemases (Brasil, 2022).

To the best of our knowledge, this is the first report on antibiotic resistance profiles, detection of β-lactamases and biofilm formation among Klebsiella spp. isolates from clinical samples of patients, healthcare workers and hospital environment of ICUs from Porto Velho-Rondonia, Brazilian Amazon. Our findings revealed that the percentage of isolates showing antibiotic resistance and MDR as well as the ability to produce biofilms, ESBL and carbapenemases was high in hospital structures and patients. These findings reinforce the urgent need to adopt rigorous disinfection measures for hospital surfaces, monitoring antibiotic resistance in pathogenic microorganisms, correct use of personal protective equipment and good infection control practices in the hospital environment, with the aim of to increase patient safety and control the spread of antibiotic-resistant bacteria in ICUs.

Acknowledgements

To the Postgraduate Program in Cellular and Molecular Biology (PGBCM) of the Oswaldo Cruz Institute (IOC), Oswaldo Cruz Rondônia Foundation (FIOCRUZ/RO), Tropical Medicine Research Center (CEPEM) and Rondônia State Health Department for their support in carrying out the project. We thank the funding institutions such as: National Institute of Epidemiology in the Western Amazon – INCT – EPIAMO, Ministry of Health; Research Program for SUS (PPSUS) and Foundation to Support the Development of Scientific and Technological Actions and Research in the State of Rondônia (FAPERO), CHAMADA 001/2018 PPSUS (011331000510000.04/2018).

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Publication Dates

• Publication in this collection
  24 Feb 2025
• Date of issue
  2024

History

• Received
  11 May 2024
• Accepted
  12 Nov 2024