Prevalence of Multidrug-Resistant Gram-Negative Bacteria From Blood Cultures and Rapid Detection of Beta-Lactamase-Encoding Genes by Multiplex PCR Assay

Abstract

Introduction: This study aimed to determine the prevalence of multidrug-resistant Gram-negative bacteria (GNB) from blood cultures in a tertiary-care hospital and the multiplex PCR assay’s ability to detect resistance genes. Methods: A total of 388 GNB isolates obtained from hospitalized patients between November 2019 and November 2021 were included in the study. Antimicrobial susceptibility testing was done by VITEK 2 system and broth microdilution method. Beta-lactamase-encoding genes were detected by multiplex PCR assays, BioFire-Blood Culture Identification 2 (BCID2) panel (bioMérieux, France). Extended-spectrum beta-lactamases (ESBLs) were detected phenotypically with VITEK AST-GN71 card (bioMérieux, France). The isolates of GNB were classified into multidrug-resistant, extensively-drug-resistant, and pandrug-resistant categories, and their prevalence and distribution in different wards, including coronavirus diseases 2019 (COVID-19) intensive care units (ICU), were calculated. Results: Results revealed that all isolates of Acinetobacter baumannii and Pseudomonas aeruginosa were multidrug-resistant as well as 91.6% of Enterobacter cloacae, 80.6% of Proteus mirabilis, and 76.1% of Klebsiella pneumoniae, respectively. In fermentative bacteria, bla_OXA-48-like (58.1%), bla_NDM (16.1%), bla_KPC (9.7%) and bla_VIM (6.5%) genes were detected. More than half of Enterobacter cloacae (58.3%) and Klebsiella pneumoniae (53.7%) produced ESBLs. Among non-fermenters, the bla_NDM gene was carried by 55% of Pseudomonas aeruginosa and 19.5% of Acinetobacter baumannii. In the COVID-19 ICU, Acinetobacter baumannii was the most common isolate (86.1%). Conclusions: This study revealed high proportions of multidrug-resistant blood isolates and various underlying resistance genes in Gram-negative strains. The BCID2 panel seems to be helpful for the detection of the most prevalent resistance genes of fermentative bacteria.

Introduction

Increased antibiotic resistance has become a global health issue, limiting options to prevent and treat bacterial infections. Antibiotic resistance increases mortality rates, lengthens hospital stay, and drives up medical costs [1]. The mortality rate caused by resistant strains of Gram-negative bacteria (GNB) is extremely high, especially in intensive care units (ICU) [2]. Since beta-lactam antibiotics are the most commonly prescribed antimicrobials, resistance typically develops early. The hydrolysis by beta-lactamases is the commonest resistance mechanism, and the most prevalent enzymes include extended-spectrum beta-lactamases (ESBLs) and carbapenemases [3]. The clinically very important groups of ESBLs are CTX-M enzymes, followed by SHV and TEM-derived enzymes, which degrade penicillins, cephalosporins, and aztreonam but not cefamycins [3]. Many studies have found that patients with bacteremia caused by ESBL-producing strains of Enterobacterales have a high mortality rate [4]. The most important carbapenemase enzymes are Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM), Verona integron-encoded metallo-beta-lactamase (VIM), imipenemase (IMP), and oxacillinase (OXA)-type, which are capable of hydrolyzing the most powerful drugs, including carbapenems [3]. Bloodstream infections (BSI) caused by multidrug-resistant (MDR) GNB strains are serious infections, leading to septic shock as a life-threatening condition, with ICU patients at the greatest risk [5]. The highest overall antibiotic resistance in the ICU, in a public hospital in Greece, was reported for Acinetobacter spp. (93%), followed by Klebsiella spp. (72.3%) and Pseudomonas spp. (49%) [6]. In a study conducted in Italy, Enterobacterales accounted for 88.7% of all BSIs, of which 21.9% were MDR [7]. It also appears that patients with coronavirus disease 2019 (COVID-19) patients have been more sensitive to ICU-acquired infections [8]. So, the current study aimed to estimate MDR GNB in our medical center, which provides tertiary health services to a population of approximately two million people in the central Serbia region. The obtained data from this research might further provide a baseline for future comparative studies.

Methods

The study was designed as a cross-sectional study conducted from November 1, 2019, to November 30, 2021, at the University Clinical Center (UCC) Kragujevac. The Ethics Committee of the UCC Kragujevac, Serbia (reference number 01/22-23; January 24, 2022) approved this study. According to the Ethics Committee, no informed consent was required for the collection, analysis, and publication of this data, because all patients were deidentified and clinical samples re-coded.

Isolation and identification of GNB from blood cultures

Bacterial isolates from blood cultures were obtained in the Microbiology laboratory at UCC Kragujevac as part of routine work using an automated blood culture system, Bact/ALERT (bioMérieux, France). Bacterial identification of GNB was done by the VITEK 2 system (bioMérieux, France) and conventional bacteriological techniques, such as a colony appearance on sheep blood agar and MacConkey agar, Gram-staining, motility tests, and biochemical characteristics. GNB were obtained from 675 blood samples, and 388 non-repetitive GNB isolates (one per infected patient) were included in further research.

Antimicrobial susceptibility testing and the detection of ESBLs

The minimum inhibitory concentrations (MICs) to ampicillin, amoxicillin-clavulanic acid, piperacillin-tazobactam, ceftriaxone, cefepime, ceftazidime, imipenem, meropenem, ertapenem, ciprofloxacin, trimethoprim-sulfamethoxazole, gentamicin, amikacin, and tetracycline, were determined by a VITEK 2 system (bioMérieux, France), with VITEK AST-GN71 and VITEK AST-GN72 card. MICs for colistin were evaluated by ComASP Colistin (Liofilchem, Italy). MICs for tigecycline were assessed by the broth microdilution method with Mueller-Hinton broth (Bio-Rad, UK). The susceptibility categories were evaluated following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [9,10]. For all species tested to tigecycline, we used breakpoints for Enterobacterales (E. coli, S≥0.5). A meropenem-resistant isolate (MIC>8 μg/mL) is referred to as a carbapenem-resistant (CR) isolate. For control of antimicrobial susceptibility testing, we used reference strains P. aeruginosa ATCC 27853 and E. coli ATCC 25922 to control. Additionally, we detected all ESBLs phenotypically with VITEK AST-GN71 card, which interprets the results as a positive or negative confirmatory test. The test was performed according to the manufacturer’s instructions.

Detection of beta-lactamases-encoding genes by multiplex PCR assay

All included samples were tested for the presence of acquired carbapenemase genes (bla_KPC, bla_OXA-48-like, bla_NDM, bla_IMP, bla_VIM) and ESBL genes (bla_CTX-M) with multiplex PCR test, the BioFire-Blood Culture Identification 2 (BCID2) panel (bioMérieux, France) which can identify 33 pathogens and the 10 most common resistance genes associated with bloodstream infections (two minutes of hands-on time and turnaround time of about one hour) [https://www.biomerieux-diagnostics.com/biofire-bcid-panel]. The test was performed according to the manufacturer’s instructions. However, this test cannot detect all beta-lactamase genes, which limits this research. Additionally, the concordance between conventional identification of GNB and identification by the BCID2 panel was analyzed.

Classification of isolates

The isolates of GNB were classified as follows: MDR (resistant to at least one agent in three or more antimicrobial categories), extensively-drug resistant (XDR) (resistant to at least one agent in all but two or fewer antimicrobial categories), and pandrug-resistant (PDR) (resistant to all agents in all tested antimicrobial categories) [11]. XDR and PDR isolates were considered subsets of MDR isolates. The prevalence of MDR isolates in different wards, including COVID-19 ICU, was calculated.

Results

Overall, the results revealed that the most common GNB species were A. baumannii (195/388; 50.3%), followed by K. pneumoniae (67/388; 17.3%), E. coli (50/388; 12.9%), P. mirabilis (31/388; 8.0%), and P. aeruginosa (27/388; 7.0%), respectively. On the other hand, other species had a lower prevalence [Enterobacter cloacae (12/388; 3.1%), Salmonella spp. (3/388; 0.8%), Providencia rettgeri (2/388; 0.5%), and Serratia marcescens (1/388; 0.3%), respectively]. The GNB identification by the BCID2 panel was consistent with the identification by the conventional method in 386/388 samples to the genus level (the panel had no target for Providencia spp.).

Antimicrobial resistance

The antibiotic resistance patterns of Gram negative (GN)-fermenting isolates are shown in

Table 1. Antimicrobial resistance of Gram-negative fermenting bacteria blood isolates.

. Among GN-fermenting bacteria, resistance to third-generation cephalosporins and aminopenicillins was very high, ranging from 33.3% to 100%, depending on the species (Table 1). Resistance to gentamicin was detected in 50% to 71% of the isolates of K. pneumoniae, P. mirabilis, and E. cloacae, while amikacin proved to be more effective, with resistance rates of 17.9-33.3%. Four isolates of K. pneumoniae were resistant to meropenem and ertapenem and susceptible to imipenem, while 23.9% were resistant to all three of them. Only two isolates of K. pneumoniae were resistant to colistin (3%). The resistance of colistin was not found in any other strains. Only one E. coli isolate was resistant to piperacillin-tazobactam and carbapenems. Other details are shown in Table 1. Among non-fermenting bacteria, all A. baumannii isolates were CR, as well as 77.8% of P. aeruginosa (

Table 2. Antimicrobial resistance of Gram-negative non-fermenting bacteria blood isolates.

). Only three isolates of A. baumannii were resistant to colistin and had a MIC >2 μg/mL (Table 2). More details are shown in Table 2.

Prevalence of MDR, XDR and PDR isolates

There were in total 334 MDR isolates. The percentage of MDR within each species is shown in Figure 1. All A. baumannii and P. aeruginosa were MDR. MDR isolates also were predominant (76.1-91.6%) in K. pneumoniae, P. mirabilis, and E. cloacae. All A. baumannii isolates at the same time were XDR, as well as 77.8% of P. aeruginosa and 29.9% of K. pneumoniae. XDR phenotype was found in one (2%) E. coli and five (16.1%) P. mirabilis. A total of three isolates (1.5%) of A. baumannii were PDR (Figure 1).

MDR phenotypes and the distribution of GNB species in various hospital wards

The most common MDR phenotype was MDR phenotype IV, which included resistance to carbapenems (58.4% of A. baumannii, 6.3% of P. aeruginosa and 6% of K. pneumoniae, respectively) followed by MDR phenotype III [susceptibility to carbapenems and resistance to other beta-lactams, fluoroquinolones, and aminoglycosides] (5.1% of K. pneumoniae and 5.1% of P. mirabilis) (Figure 2a). Other phenotypes are shown in Figure 2a.

The highest prevalence of GNB was recorded in the ICUs, both COVID-19 ICU (122/388; 31.4%) and surgical ICU (97/388; 25.0%), as well as in the emergency center (63/388; 16.2%). In other wards it was lower, [internal (27/388; 7%), urology and nephrology (23/388; 5.9%), oncology (23/388; 5.9%), infectious disease (18/388; 4.6%) and pediatric ward (15/388; 3.9%), respectively]. The distribution of MDR phenotypes in the COVID-19 ICU is displayed in Figure 2b. Among these, A. baumannii was the most common isolate (86.1%). All above mentioned isolates recovered from COVID-19 ICU were CR (MDR phenotype IV). Other phenotypes have been very rare (0.8%) (Figure 2b).

Evaluation of beta-lactamase-encoding genes detection and ESBLs production

Among CR GN-fermenting bacteria, carbapenemase-encoding genes were detected in 87.1% (27/31) (

Table 3. The frequency of the isolates with the beta-lactamase-encoding genes.

). The most prevalent were bla_OXA-48-like (18/31; 58.1%) and bla_NDM (4/31; 12.9%), followed by bla_KPC (3/31; 9.7%) and bla_VIM (2/31; 6.5%). Detection of the carbapenemase-encoding genes in non-fermenting GNB by the BCID2 panel was successful in 17/21 (81.0%) of CR P. aeruginosa (55% bla_NDM and 28.6% bla_VIM) and 38/195 (19.5%) of A. baumannii isolates (only bla_NDM detected). None of the isolates showed presence of bla_IMP. More details are shown in Table 3. The most common ESBL genes, bla_CTX-M, were found in 15/67 (22.4%) K. pneumoniae, 5/50 (10%) E. coli, and 1/12 (8.3%) E. cloacae isolates, respectively. The prevalence of all ESBL producing isolates, obtained by VITEK 2 AST-GN71, was as follows: 58.3% (7/12) for E. cloacae, 53.7% (36/ 67) for K. pneumoniae, 30% (15/50) for E. coli, and 25.8% (8/31) for P. mirabilis.

Discussion

Until the last decade, healthcare-associated primary BSI was one of the six most prevalent infections [12]. According to the European Antimicrobial Resistance Surveillance Network (EARS Net) and the World Health Organization (WHO) data, E. coli and Klebsiella spp. represented the majority of GNB blood culture isolates in Europe over the last two years [13,14,15].

Similar to our finding, the WHO reported the greatest prevalence of MDR K. pneumoniae (more than 50%) in Eastern European countries, while Spain, Italy, and France had 10% to 25% [13,14,15]. On the other hand, the prevalence of MDR E. coli at UCC Kragujevac was 44%, more than it was reported from Italy, Cyprus, and Slovakia (approximately 10%) [13,14,15]. The presence of ESBL was found in 53.7% of K. pneumoniae and 30% of E. coli, while the bla_CTX-M gene was confirmed in 22.4% of K. pneumoniae and 10% of E. coli in UCC Kragujevac, which is probably the reason for the high prevalence of MDR isolates. Although they were among the first discovered ESBLs, the importance of TEM-type and SHV-type has decreased in recent decades due to the spread of CTX-M beta-lactamases [3]. However, since bla_CTX-M was confirmed in our institution in only one-third of the isolates compared to the total number of phenotypically detected ESBLs, it is assumed that genes for other ESBLs, such as bla_TEM and bla_SHV-type, are still significantly represented.

One of the CR E. coli was XDR (2%), similar to reports from North Macedonia, Spain, Turkey, and Ukraine, which had more than 1% of CR E. coli recently [13,14,15]. According to the “European Survey of Carbapenemase-Producing Enterobacteriaceae (EuSCAPE)” study, conducted 2013-2014, OXA-48-like was the most prevalent carbapenemase in E. coli (56%), followed by NDM (26%), and KPC (18%) [16]. The CR E. coli isolate from our hospital carried the bla_OXA48 gene, which is the most common in Europe, but it is interesting that Italy is one of the few countries where KPC E. coli isolates appeared [17]. KPC-producing E. coli was detected only in Israel, Italy, Portugal, Greece, and Cyprus [16]. In Italy, 30 distinct KPC-producing E. coli strains were identified between 2010 and 2018 [17].

Our results showed that all fermentative bacterial isolates with MICs to meropenem greater than 8 μg/mL, also produced ESBLs. In CR isolates of K. pneumoniae, bla_CTX-M genes were simultaneously detected in 15/20 (75%) isolates with bla_OXA-48 genes, in 3/20 (15%) with bla_KPC, and in 2/20 (15%) with bla_NDM genes. Also, bla_OXA-48 and bla_CTX-M genes were found together in one E. cloacae isolate. Other ESBL positive isolates were susceptible to meropenem. The first KPC isolate in the Molise region, Central Italy, carried the bla_KPC and bla_TEM genes [17].

A bla_OXA-48-like gene was found in more than half of the GN-fermenting isolates in our hospital (58.1%), which is not surprising considering that over the past decade, many European countries, especially in the Mediterranean area, have reported a rapid spread of strains that produce OXA-48 carbapenemases [18].

Enterobacter spp. also increased the global number of ESBL-positive and CR strains. For example, in Arab countries over the last 20 years, CR Enterobacter spp. ranged between 29.4-30.6% and resistance to third-generation cephalosporins was more than 60% [19]. We found only three CR E. cloacae with different types of carbapenemase-encoding genes (bla_OXA-48-like, bla_NDM, and bla_VIM), but 58.3% of isolates were ESBL positive. The increasing prevalence of MDR P. mirabilis, especially those that produce ESBLs, has been recorded all over the world as well as at our institution (five isolates were CR and 25.8% produced ESBLs) [20]. The most successful antibiotics in vitro against fermenting bacteria except colistin and carbapenems were piperacillin-tazobactam (resistance rate of 2-33.3%), amikacin (17.9-33.3%), and tigecycline (0-23.9%).

The spread of resistant strains of A. baumannii and P. aeruginosa is a global problem worldwide. A study in Spain confirmed high mortality (20-33% within the first 5 and 30 days) in patients with BSI caused by P. aeruginosa (38.3% of isolates were MDR while 93.5% were XDR) [21]. Furthermore, the ten-year prevalence study in Brazil (2006–2016) showed that 76.8% of A. baumannii were XDR [22]. In our hospital, all A. baumannii isolates were XDR, as well as 77.8% of P. aeruginosa. The appearance of XDR strains that are resistant to colistin, even though there were only five of them in our study is alarming because colistin is considered the last line of defense and a reserve antibiotic [23].

Among CR GN-non-fermenting isolates, our study detected more A. baumannii bla_NDM gene carriers (19.5%), compared to data from Serbia in 2018, where just 3.2% of A. baumannii carried the bla_NDM gene [24]. This raises the assumption that Serbia might be an endemic region for CR isolates carrying bla_NDM genes. According to that research, among the tested CR A. baumannii isolates in Serbia, bla_OXA-24 was the most frequent carbapenemase gene (44.2%), followed by bla_OXA-23 (34.5%), but the BCID2 panel does not have the ability to detect these genes [24]. In the P. aeruginosa strains, bla_NDM (55%) and bla_VIM (28.6%) genes were detected.

The majority of GNB blood isolates at UCC Kragujevac were from critical care units: COVID-19 ICU (31.4%), surgical ICU (25.0%), and emergency center (16.2%), which is expected, because patients in ICUs are the most sensitive to bacterial infections everywhere in the world, as mentioned above [2,5,8]. Patients with severe COVID-19, according to multiple researchers, have a higher rate of secondary infections than patients with non-severe COVID-19 [25]. In the COVID-19 ICU at UCC Kragujevac, 86.1% of all GNB isolates were CR A. baumannii. Other GNB have also been MDRs, with the majority of isolates resistant to carbapenems, which is similar to other tertiary care institutions worldwide [25].

The limitation of the present research could be that this was a single medical center-based study. Further, we mention the restriction of multiplex PCR on GNB genetic resistance detection including beta-lactamase-encoding genes (the test does not have targets for all beta-lactamase-encoding genes, particularly in non-fermentative bacteria) and other resistance mechanisms.

Conclusions

The fact that all of the GN-non-fermenting isolates from blood cultures in our facility were MDR as well as the majority of GN-fermenting bacteria (more than 70% of K. pneumoniae, P. mirabilis, and E. cloacae) raise serious concern. Our results show a high proportion of CR-resistant strains of GNB, probably due to the spread of carbapenemase-encoding genes, particularly bla_OXA-48-like and bla_NDM. CR A. baumannii was the most commonly isolated GNB in the last year, especially in the COVID-19 ICU, because it spreads rapidly in hospital conditions and in critically ill patients. All isolates of P. aeruginosa, K. pneumoniae, and E. cloacae from the COVID-19 ICU were also CR, so the multiplex PCR test, with rapid detection of resistance genes, enabled the application of adequate therapy before the results of antimicrobial testing have been done. It has been shown that the multiplex PCR test, BioFire BCID2 panel, can be helpful for rapid detection of the most common resistance genes, especially for GN-fermenting bacteria, but further development of this test for the detection of other OXA-carbapenemases is needed. Future efforts must include urgent research into novel antibiotics and the implementation of preventive measures in the hospital and community environment to prevent the spread of MDR bacteria.