The number of bacterial isolates extremely resistant to previously effective drugs is growing dynamically. Infections are increasingly being caused by pathogens that are not susceptible to all available antibiotics. This issue is particularly acute for Gram-negative bacilli, both the Enterobacterales strains and non-fermenting rods. Colistin is used as one of the last available treatment options for patients with severe infections caused by carbapenem-resistant Gram-negative rods. Due to the increasing role of colistin in the treatment of human infections caused by multidrug-resistant (MDR) bacteria, the resistance to this antibiotic ought to be monitored (Prim et al. 2017; Petrosillo et al. 2019; Stefaniuk and Tyski 2019).
Until recently, colistin resistance was thought to be dependent only on mutations in the genes regulating LPS synthesis. In 2015, the plasmid-coded colistin resistance associated with the presence of
The twelve hospitals located all over Poland, in the following voivodeships: Lesser Poland (n = 2), Lublin (n = 1), Masovian (n = 2), Pomeranian (n = 1), Silesian (n = 2), Warmia-Masurian (n = 2), and West Pomeranian (n = 2), involved in this study were of similar sizes and had similar profiles, as regional, secondary-care medical centers, with all major types of wards. A total of 65 non-duplicate clinical isolates of Enterobacterales were recovered from inpatients with various infections between April 2019 and December 2019 and were included in this study. The strains were of the following species:
Based on the information provided by the laboratories, the results of antibiotic susceptibility of the studied bacterial strains were pre-analyzed. In the NMI, the colistin MIC value (mg/l) was determined by a reference broth microdilution method according to ISO 20776 (ISO 2019). Susceptibility to colistin was performed in triplicate for each strain, using the same culture to establish a pool of strains with MIC > 2 mg/l of colistin. E-tests with concentration gradients of ceftazidime, ceftazidime/avibactam, imipenem, meropenem, and meropenem/vaborbactam (MIC Strep; Liofilchem, Italy) were used for determination of their MICs (mg/l) in colistin-resistant Enterobacterales strains. Susceptibility results were interpreted according to the guidelines of the EUCAST (EUCAST 2020a). The following strains:
All Enterobacterales isolates were tested for ESBLs and carbapenemases production by phenotypic and genotypic methods. ESBLs were detected by the double-disk synergy (DDS) test with disks containing amoxicillin with clavulanate (20 μg and 10 μg, respectively), cefotaxime (30 μg), and ceftazidime (30 μg) (EUCAST 2017). The detection of carbapenemases were assessed by the disk test with phenylboronic acid for KPCs, the synergy test with EDTA for MBLs, and disc with temocillin for OXA-48-like carbapenemases (Żabicka et al. 2015).
Total bacterial DNA was purified with a Genomic DNA Prep Plus kit (A&A Biotechnology, Gdańsk, Poland).
The blaCTX-M-1-, blaCTX-M-2-, blaCTX-M-8- blaCTX-M-9-, blaCTX-M-25-, blaSHV-, blaTEM- blaKPC-, blaNDM-, blaIMP-, blaVIM-, blaOXA48-like genes were identified by PCR as described previously (Woodford et al. 2006; Empel et al. 2008).
All isolates were screened by PCR for the presence of plasmid-mediated
The isolates came from patients of various ages from 1 to 89 years; the most numerous group comprised of patients aged 61–80 (n = 32; 49.2%) and 31–60 years of age (n = 16; 24.6%). The remaining patients were 16–30 years of age (n=4), ≥ 81 years of age (n=11), and <3.1 years (n=2). The most frequently represented hospital wards were: Intensive Care Unit (n = 20, 30.8%), internal medicine (n = 14, 21.5%), pulmonary (n = 12; 18.5%), and burn wards (n = 8; 12.3%). The remaining patients were hospitalized in the following order: surgery (n = 3), rehabilitation (n = 3), urology (n = 1), and oncology (n = 1). Three patients from whom the tested strains were isolated were patients of the surgical outpatient clinics (n = 2) and one resident of the Long Term Care Facility with documented hospital history.
Just over 40% of all patients’ clinical specimens (n = 28; 43.1%) for microbiological testing came from the lower respiratory tract, including: bronchial lavage (n = 16; 24.6%), and sputum (n = 11; 16%), pleural fluid (n = 1), specimens from skin and soft tissue infections (n = 8, 12.3%), and urine (n = 13, 20%). Only 16.9% (n = 11) of the Enterobacterales isolates tested were collected from blood; single isolates came from peritoneal fluid (n = 1), bile (n = 1), and rectal swabs (n = 3).
Resistance to colistin was demonstrated in all 65 isolates. The MIC values of colistin in resistant strains ranged from 4 mg/l to > 64 mg/l. For PCR, positive results were achieved only with primers specific to the
Susceptibility of colistin-resistant Enterobacterales strains (n = 65) to ceftazidime, ceftazidime/avibactam, imipenem, meropenem, and meropenem/vaborbactam.
Strains (n; %) | Colistin | CAZ | CAZ/AVB | IPM | MEM | MEM/VB | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
MIC (mg/l) | S | R | S | R | S | I | R | S | I | R | S | R | |||
Value | Numer of isolates | ||||||||||||||
4 | 11 | 0 | 0 | 11 | 9 | 2 | 9 | 0 | 2 | 9 | 1 | 1 | 10 | 1 | |
8 | 4 | 0 | 4 | 0 | 4 | 0 | 4 | 0 | 0 | 4 | 0 | 0 | 4 | 0 | |
16 | 2 | 0 | 0 | 2 | 1 | 0 | 2 | 0 | 0 | 2 | 0 | 0 | 2 | 0 | |
32 | 1 | 0 | 1 | 0 | 1 | 1 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | |
64 | 1 | 0 | 1 | 0 | 1 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | |
> 64 | 26 | 0 | 14 | 12 | 24 | 2 | 19 | 1 | 6 | 19 | 2 | 5 | 23 | 3 | |
4 | 4 | 0 | 2 | 2 | 4 | 0 | 4 | 0 | 0 | 4 | 0 | 0 | 4 | 0 | |
8 | 5 | 0 | 3 | 2 | 5 | 0 | 5 | 0 | 0 | 5 | 0 | 0 | 5 | 0 | |
16 | 3 | 1 | 0 | 3 | 3 | 0 | 3 | 0 | 0 | 3 | 0 | 0 | 3 | 0 | |
32 | 1 | 1 | 0 | 1 | 0 | 1 | 0 | 0 | 1 | 0 | 1 | 0 | 1 | 0 | |
64 | 1 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | |
> 64 | 1 | 0 | 0 | 1 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 1 | 0 | |
4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
8 | 1 | 0 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | |
16 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
32 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
64 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
> 64 | 2 | 0 | 1 | 1 | 2 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 2 | 0 | |
4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
8 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
16 | 1 | 0 | 1 | 0 | 1 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | |
32 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
64 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
> 64 | 1 | 0 | 1 | 0 | 1 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | |
Total | 65 | 3 | 28 | 37 | 59 | 6 | 52 | 3 | 10 | 53 | 5 | 7 | 61 | 4 |
CAZ – ceftazidime, CAZ/AVB – ceftazidime/avibactam, IPM – imipenem, MEM – meropenem, MEM/VB – meropenem/vaborbactam, S – sensitive, I – intermediate, R –resistant
In Kazmierczak and co-researcher’s study (2018) the most common ESBL genes in Polish isolates was CTX-M-15 (80% of 185 ESBL-positive isolates). Authors also observed high percentages of MDR Polish strains (21%); 29.2% of them were ceftazidime-resistant and 0.8% meropenem non-susceptible, but only one isolate produced carbapenemase and it belonged to carbapenemase subtype VIM-1. A higher percentage of Enterobacterales strains resistant to ceftazidime (56.9%) and non-susceptible to meropenem (16.9%) was observed in our study.
Forty-five of the colistin-resistant isolates (69.2%) were identified as ESBL producers by the DDS test. The ESBL-positive strains belonged to three species including
One
The results of the detection of selected β-lactamases are shown in Table II.
Presence of selected ESBLs and carbapenemases among colistin-resistant Enterobacterales strains (n = 65*).
Strains (n; %) | Colistin | Types of ESBLs | Types of carbapenemases | |||||||
---|---|---|---|---|---|---|---|---|---|---|
MIC (mg/l) | CTX-M-1 | CTX-M-9 | TEM | SHV | KPC | NDM | OXA-48 | |||
Value | Number of isolates | |||||||||
4 | 11 | 0 | 6 | 1 | 2 | 0 | 0 | 2 | 0 | |
8 | 4 | 0 | 4 | 0 | 0 | 0 | 0 | 0 | 0 | |
16 | 2 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | |
32 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | |
64 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | |
> 64 | 26 | 0 | 22 | 1 | 3 | 4 | 1 | 3 | 1 | |
4 | 4 | 0 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | |
8 | 5 | 0 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | |
16 | 3 | 1 | 1 | 0 | 1 | 1 | 0 | 0 | 1 | |
64 | 1 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | |
8 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | |
> 64 | 2 | 0 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | |
Total | 61 | 3 | 41 | 2 | 10 | 5 | 1 | 5 | 2 |
– in two
The growing resistance of bacteria to antibiotics is a challenge for 21st-century medicine. Carbapenems were considered so-called “last resort” agents in the treatment of serious infections, especially in hospitalized patients. The spread of carbapenem-resistant Gram-negative rods isolated from outpatients turned out to be a challenge for treating infections (Grundmann et al. 2010; Parisi et al. 2015). The expansion of strains producing carbapenemases has been observed for several years worldwide, including in Poland (Baraniak et al. 2016). Numerous reports have indicated the disturbing phenomenon of large-scale spreading of Enterobacterales strains producing New Delhi metallo-β-carbapenemase, and to a lesser extent producing
Colistin is characterized by high activity against Gram-negative rods, despite numerous reports of increasing bacterial resistance to this drug (Petrosillo et al. 2019), most of which are chromosomally coded. The spread of plasmid-encoded resistance to colistin, related to the presence of
The project achieved the collection of colistin-resistant Enterobacterales rods over three quarters of 2019. Within the total number of collected strains, isolates with the
Globally, the
This is the first report on the occurrence of β-lactamases in colistin-resistant Enterobacterales strains in Poland. These data broaden the knowledge of the mechanism of resistance to colistin among Enterobacterales causing human infections in Poland. Demographic data of patients, from whom the strains resistant to colistin were isolated, indicate that the problem of this resistance cannot be limited to a selected group of patients. The small number of colistin-resistant isolates (n = 65) obtained from hospitals that participated in the pilot study may indicate that the problem of colistin resistance among Enterobacterales strains is low. However, due to the described issues of the infection therapy, this problem requires further research and analysis. In the future, the authors plan to compare the antibiotics susceptibility of Enterobacterales isolates resistant to colistin and other multidrug-resistant Enterobacterales species susceptible to colistin.