In addition, it has developed high resistance to many antibiotic classes (4, 5) through its defence mechanisms, which involve reducing antibiotic concentration in the cell, changing the target site, and antibiotic inactivation by its own or acquired enzymes. Multidrug resistance (MDR) of
Biofilm formation is a complex process that begins with the binding of bacteria to the surface, after which they multiply, aggregate and form multilayer deposits, which could serve as a reservoir for the spreading of living cells and cause the development of chronic infections.
Since
The aim of our study was to determine the frequency of multidrug-resistant
Between 2013 and 2015, we collected 94 bacterial isolates, initially characterised as
The obtained samples were inoculated within a few hours on the following substrates: Columbia blood agar base, MacConkey agar, endo agar, tryptone soy agar, Mueller-Hinton agar (MHA), glucose, and thioglycollate broths (all purchased from Oxoid Ltd., Basingstoke, UK). Luria-Bertani (LB) medium and cetrimide agar (Lab M Limited, Bury, UK) were used for further testing. All isolates were grown overnight at 37 °C, stocked in 30 % glycerol, and stored at -80 °C until further use.
The use of clinical strains was approved by the hospital’s ethics committee (decision No. 3242). Patient-identifiable information was coded and hidden from us and we had no contact with patients who gave the samples, so no informed consent was necessary for this study. None of the clinical data previously obtained were associated with the isolates of this study.
Pigment production of
Biofilm formation was quantified with a modified version of the method described by Stepanović et al. (16). Each isolate was tested in triplicate (in three wells of 96-well microtiter plate), and all tests were carried in three separate experiments during three consecutive days. The wells of a 96-well flat-bottomed microplate were filled with a final volume of 200 μL [180 μL of Mueller-Hinton broth (MHB) and 20 μL of bacteria (5×105 CFU/mL)]. Negative control wells contained MHB only. After incubation at 35 °C for 24 h, the plates were decanted and the wells washed three times with 300 μL of phosphate buffer (1×PBS, pH 7.2, 25 °C). The remaining attached bacteria were fixed with 150 μL of methanol per well, and the plates emptied after 20 min and left to air dry. The final step was staining with 150 μL of 2 % Crystal Violet (Lach-Ner, Neratovice, Czech Republic) per well for 15 min. Excessive stain was rinsed off with running tap water. After air drying, the remaining stain was solved with 150 μL of 96 % ethanol per well. Forty-eight hours later, we measured biofilm optical density (OD) of each strain with an automated Multiskan FC reader (Flow Titertek Multiskan Plus, Flow Laboratories Co., Turku, Finland) at 570 nm. The obtained readings served to set the OD cut-off values (ODc) (three standard deviations above the average mean OD of negative control) as described elsewhere (16). Each strain was classified into one of the following categories: not a biofilm producer (OD≤ODc, category 0 or -); weak biofilm producer (OD≤ODc≤2×ODc, category 1 or +), moderate biofilm producer (2×ODc≤OD≤4×ODc, category 2 or ++), or strong biofilm producer (4×ODc≤OD, category 3 or +++).
Antimicrobial susceptibility of the isolates was tested with the disk diffusion method following the protocol described by the Clinical and Laboratory Standards Institute (17). For this purpose we used standardised single antibiotic discs (Rosco Diagnostica, Taastrup, Denmark) at the following concentrations (μg per disc): 5 μg for ofloxacin, ciprofloxacin, and levofloxacin; 10 μg for piperacillin/tazobactam, imipenem, doripenem, meropenem, colistin, aztreonam, gentamicin, and tobramycin; and 30 μg for ceftazidime, cefepime, netilmicin, and amikacin. Readings were taken after incubation at 37 °C for 24 h and expressed in mm. The isolates were classified as susceptible (S), intermediate (I) or resistant (R).
The sensitivity of our
The production of ESBLs was detected with the double-disk synergy test (DDST) and confirmed with the combination disk test (CDT) (20). For DDST, the discs of amoxicillin/clavulanic acid (AMC, 20/10 μg), cephalexin (CL, 30 μg), and cefotaxime (CTX, 30 μg) were placed on plates inoculated with
Carbapenemase production was tested with a modified Hodge test (21). An overnight culture of
The isolates were tested for MBL production with the EDTA synergy test as described elsewhere (22). Briefly, an overnight liquid culture of the tested isolate (turbidity adjusted to 0.5 per McFarland standard) was spread on the surface of the MHA plate. Two imipenem discs (10 μg) were placed on the agar 15 mm apart and 10 μL of 0.5 EDTA was pipetted on one of the imipenem discs. After an overnight incubation at 37 °C, the expanded inhibition zone between the two discs or expansion of more than 6 mm in the imipenem/EDTA disc were interpreted as positive for MBL production.
Genomic DNA (gDNA) was isolated from the
The ERIC and BOX PCR reactions were done in the following steps: initial denaturation at 95 °C for 7 min, 30 cycles of denaturation at 94 °C for 1 min, primer annealing 46 °C for 1 min, and polymerisation at 65 °C for 8 min. For the 272 primer the steps were as follows: initial denaturation at 94 °C for 2 min, 35 cycles of denaturation at 94 °C for 30 s; primer annealing at 35 °C for 30 s, and polymerisation at a 72 °C for 2 min. The steps for the 208 primer were: four cycles of auto-extension (each consisting of initial denaturation at 94 °C for 5 min, primer annealing at 36 °C for 5 min, and polymerisation at 72 °C for 5 min), 30 cycles of denaturation at 94 °C for 1 min; primer annealing at 36 °C for 1 min, and polymerisation at 72 °C for 2 min. The final extension step for BOX and ERIC was done at 65 °C for 16 min and for 208 and 272 primers at 72 °C for 10 min.
From the obtained DNA fingerprints we built a dendrogram using the PyElph 1.4 software (26) and then determined clustering patterns using the unweighted pair group method with arithmetic mean (UPGMA) algorithm with a bootstrap value of 100. The position of pattern strips was checked manually.
The total DNA of selected isolates was used for 16S rRNA PCR amplification and isolate identification with universal primers UN116sF (GAGAGTTTGATCCTGGC) and UN116sR (AGGAGGTGATCCAGCCG). The reaction mixture was prepared as described above, and the conditions were the usual ones for primer annealing at 50 ºC for 1 min. The amplicons were purified on a QIAquick Gel Extraction KIT/250 column (QIAGEN GmbH, Hilden, Germany) and sequenced commercially (Macrogen, Amsterdam, Netherlands). The obtained sequences were searched for homology at the National Center for Biotechnology Information using the Basic Logical Alignment Search Tool, aligned with the ClustalW multiple sequence alignment in program BioEdit 7.1.3 (Tom Hall, North Carolina State University, Raleigh, NC, USA) and checked manually. The phylogenetic tree was constructed with the MEGA 7.0 software (Pennsylvania State University, Philadelphia, PA, USA) using the neighbour-joining method based on a pairwise distance matrix obtained with the Kimura two-parameter nucleotide substitution model. The topology of the trees was evaluated with the bootstrap resampling method with 1000 replicates.
The data obtained in this study were analysed with descriptive statistics using IBM SPSS Statistics for Windows, version 25.0 (IBM Corp., Armonk, NY, USA).
Of the 94
Table 1 shows our findings in terms of isolate serotypes, pigmentation, and biofilm formation. Twenty-eight isolates were non-typeable. All of the remaining identified serotypes were from polyvalent groups (PMA, PME, PMC, and PMF). Most belonged to the PMA and PMF groups, which included the P1 and P6 serotypes. The PMC group included the P9 and P10, while the PME and PMF groups P5 and P11 serotypes, respectively. The most commonly identified monovalent serotypes were P1 (17.02 %), P6 (22.34 %), and P11 (15.96 %). Co-production of pyoverdine and pyocyanin was observed in 70 % of clinical isolates, while the production of only pyoverdine in 22.3 % and of pyocyanin in 7.4 % of the isolates. Most isolates (77.66 %) expressed some biofilm formation ability, mostly weak (53.42 %, most of them originating from inpatient wounds and outpatient urine cultures) and moderate (39.73 %, most of them originating from inpatient wounds), while only five isolates were strong biofilm producers.
Serological identification of polyvalent and monovalent serotypes, pigmentation, and biofilm formation of
Polyvalent and monovalent serotypes | Pigments type | Biofilm formation | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Isolate origin | PMA | PME | PMC | PMF | NT | Pyov+ Pyoc | Pyov | Pyoc | Cat. 0(-) | Cat. 1(+) | Cat. 2(++) | Cat. 3(+++) | ||
P1 | P6 | P5 | P9 | P10 | P11 | |||||||||
Throat | 4 | 4 | - | - | 1 | 1 | 3 | 9 | 3 | 1 | 2 | 6 | 5 | - |
Tongue | - | 1 | - | - | 1 | - | - | 1 | 1 | - | 1 | - | 1 | - |
Wound | 7 | 7 | 3 | 6 | - | 8 | 13 | 30 | 12 | 2 | 12 | 17 | 14 | 1 |
Cer-vagmal canal | - | - | - | - | - | - | 2 | 1 | 1 | - | 1 | - | 1 | - |
Ear | - | 2 | - | - | - | 1 | 1 | 2 | 1 | 1 | - | - | 4 | - |
Sputum | 1 | 4 | - | 1 | - | 1 | 2 | 7 | 1 | 1 | 2 | 4 | 1 | 2 |
Urine culture | 4 | 3 | - | 2 | - | 4 | 7 | 16 | 2 | 2 | 3 | 12 | 3 | 2 |
Σtotal isloates |
NT – non-typeable; Pyov – pyoverdine; Pyoc – pyocyanin; Cat. 0 (-) – not a biofilm producer; Cat. 1 (+) – weak biofilm producer; Cat. 2 (++) – moderate biofilm producer; Cat. 3 (+++) – strong biofilm producer
These findings are consistent with earlier reports showing dominance of the P3, P6, and P11 serotypes (28, 29, 30) and pigmentation where the synthesis of pyoverdine (79.75 %) was more pronounced than the synthesis of pyocyanin (44.14 %) (27). The pigments of the clinical
Antibiotic susceptibility of inpatient and outpatient
Inpatients | ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Isolate coded name | Specimen | Aminoglycosides | Fluoroquinolones | Polyp | Monob | Penicillin comb. | Carbapenems | Cephalosporins | ||||||||
Amik | Gent | Net | Tobr | Oflox | Cipr | Levofl | Col | Aztr | Pip/taz | Dor | Imp | Mer | Ceft | Cef | ||
5660 | Throat | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
5661 | Throat | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
1183 | Urine | S | S | S | S | S | S | S | ||||||||
2638 | Urine | S | S | |||||||||||||
2844 | Urine | S | S | S | S | S | S | S | S | S | ||||||
3853 | Urine | S | S | S | S | S | S | S | S | |||||||
611 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
821 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | |
971 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | ||
1087 | Wound | S | S | |||||||||||||
1416 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | |
2064 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | ||
2073 | Wound | S | S | S | ||||||||||||
2581 | Wound | S | S | S | S | S | S | |||||||||
2645 | Wound | S | S | |||||||||||||
2900 | Wound | S | S | S | ||||||||||||
3122 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | ||
3322 | Wound | S | S | S | S | S | S | |||||||||
3451 | Wound | S | S | S | S | S | S | S | ||||||||
3563 | Wound | S | S | S | S | S | S | S | S | |||||||
3595 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
3658 | Wound | S | S | S | S | S | S | S | S | |||||||
3714 | Wound | S | S | S | S | S | S | S | S | S | ||||||
3883 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
4071 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
4082 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
4211 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | |
4212 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
4312 | Wound | S | S | S | S | S | S | S | S | S | ||||||
4314 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
4354 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
4473 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
4541 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
5348 | Wound | S | S | S | S | S | S | S | ||||||||
5774 | Wound | S | S | S | S | S | I | S | ||||||||
5797 | Wound | S | S | S | S | S | S | S | ||||||||
6412 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
6982 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
7543 | Wound | S | S | S | S | S | ||||||||||
7698 | Wound | I | S | I | S | S | S | S | S | S | S | S | S | |||
7881 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | |
8283 | Wound | I | I | S | S | S | S | S | S | S | S | |||||
8702 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
8913 | Wound | S | S | S | S | S | S | S | S | |||||||
10195 | Wound | S | S | S | S | I | I | S | S | S | S | S | S | |||
10336 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
10708 | Wound | I | I | S | S | S | S | S | S | S | S | S | S | S | S | |
10800 | Wound | S | I | S | S | S | S | S | S | S | S | S | S | |||
11947 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
19677 | Wound | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
Outpatients | ||||||||||||||||
Isolate coded name | Specimen | Aminoglycosides | Fluoroquinolones | Polyp | Monob | Penicillin comb. | Carbapenems | Cephalosporins | ||||||||
Amik | Gent | Net | Tobr | Oflox | Cipr | Levofl | Col | Aztr | Pip/taz | Dor | Imip | Mer | Ceft | Cef | ||
2609 | Ear | S | S | S | S | S | S | S | S | S | S | S | S | S | S | |
4278 | Ear | S | I | S | S | S | S | S | S | S | S | S | S | S | S | S |
5518 | Ear | S | S | S | S | S | S | S | S | S | S | S | S | |||
7546 | Ear | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
294 | Sputum | S | S | I | S | S | S | S | S | S | S | S | S | S | S | |
2124 | Sputum | S | S | S | S | S | S | S | S | |||||||
2941 | Sputum | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
2966 | Sputum | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
3496 | Sputum | S | S | S | S | S | S | S | S | S | S | S | S | S | ||
3919 | Sputum | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
8142 | Sputum | S | S | S | S | S | S | S | S | S | S | S | S | S | S | |
9921 | Sputum | S | S | S | S | S | S | |||||||||
11838 | Sputum | S | S | S | S | S | S | S | S | S | S | S | S | S | ||
1863 | Throat | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
2005 | Throat | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
2047 | Throat | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
3238 | Throat | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
3477 | Throat | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
3540 | Throat | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
3864 | Throat | S | I | S | S | S | S | S | S | S | S | S | S | S | S | S |
4087 | Throat | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
4646 | Throat | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
9642 | Throat | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
10412 | Throat | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
2383 | Tongue | S | S | S | S | S | S | S | S | S | S | S | S | S | ||
2967 | Tongue | I | S | S | S | S | S | S | S | S | ||||||
1408 | Urine | S | S | S | S | S | S | S | S | S | ||||||
1790 | Urine | I | S | S | S | S | S | S | S | S | S | S | S | S | S | |
2285 | Urine | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
2588 | Urine | I | S | S | S | S | S | S | S | |||||||
2597 | Urine | S | S | S | S | S | S | S | ||||||||
2711 | Urine | I | S | S | S | S | S | S | S | S | ||||||
2875 | Urine | S | S | S | S | S | S | S | S | S | ||||||
3199 | Urine | S | S | S | S | S | S | S | S | S | ||||||
3214 | Urine | S | S | S | S | S | S | |||||||||
3777 | Urine | S | S | S | S | S | S | |||||||||
4188 | Urine | I | S | S | S | S | S | |||||||||
4362 | Urine | S | S | S | S | S | S | S | S | S | S | S | S | S | ||
5586 | Urine | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
8599 | Urine | S | S | S | S | S | S | S | ||||||||
10019 | Urine | S | S | I | S | S | S | S | ||||||||
10600 | Urine | S | S | S | S | S | S | S | S | S | S | S | S | S | ||
2671 | Vag. Swab | S | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
2689 | Vag. Swab | S | S | S | S | S | S | S | S | S | S | S | S | S | S |
Amik – amikacin; Gent – gentamicin; Net – netilmicin; Tobr – tobramycin; Oflox – ofloxacin; Cipr – ciprofloxacin; Levofl – levofloxacin; Col – colistin; Aztr – aztreonam; Pip/taz – piperacillin/ tazobactam; Dor – doripenem; Imip – imipenem; Mer – meropenem; Ceft – ceftazidime; Cef – cefepime. Polyp – polypeptide class; Monob – monobactams class. S – susceptible; I – intermediate susceptible; R– resistant isolates. Bolded letters represent resistant values
Susceptibility to 15 antibiotics from different classes such as aminoglycosides (amikacin, gentamicin, netilmicin and tobramycin), fluoroquinolones (ofloxacin, ciprofloxacin and levofloxacin), polypeptides (colistin), penicillin combination (piperacillin/tazobactam), monobactams (aztreonam), cephalosporins (ceftazidime and cefepime), and carbapenems (doripenem, imipenem and meropenem) was tested in all isolates. Figure 1 shows that all isolates were sensitive to colistin, and most to aztreonam (97.87 %), imipenem, and doripenem (91.49 % and 90.43 %, respectively), while 84.04 % of the isolates were susceptible to meropenem and the piperacillin/tazobactam combination. These findings single out colistin as antibiotic of choice, but only as a last resort due to its neurotoxicity (34).
Regarding ceftazidime and cefepime susceptibility, 70.21 % and 75.53 % of the isolates were sensitive to 3rd and 4th generation cephalosporins, respectively. The highest number of resistant isolates was observed in testing with aminoglycoside [gentamicin (39.36 %), netilmicin (35.10 %) and tobramycin (37.23 %)], and fluoroquinolone [ofloxacin (38.30 %), ciprofloxacin (34.04 %) and levofloxacin (32.98 %)] classes. Twenty-three of inpatient isolates were resistant to fluoroquinolone ofloxacin (46 %) and seventeen of outpatient isolates to aminoglycoside gentamicin (38.64 %). Cefepime best inhibited
MICs for ceftazidime, cefepime, and meropenem confirmed these findings and singled out meripenem as the most effective inhibitor, as 72 isolates (76.6 %) were highly susceptible to meropenem with MIC below 1 μg/mL (Table 3). The most resistant isolates were obtained from wounds, urine, and sputum.
Only two isolates – one from inpatient wound and the other from outpatient urine – produced ESBL, which is the likely reason for resistance to piperacillin/tazobactam, cephalosporins, and aztreonam (Tables 2 and 3). Eight isolates – five wound and three urine – produced carbapenemases. All of these inpatient wound isolates were resistant to carbapenems, and all isolates showed multidrug resistance, except 10800 (Table 2). Five isolates produced MBLs (three from inpatient wounds and two from outpatient ear and urine specimens) (Table 3). Two wound isolates were resistant to the piperacillin/tazobactam combination, while all isolates were susceptible to doripenem (Table 2).
Minimum inhibitory concentration (MIC, expressed in μg/mL) of inpatient and outpatient
Inpatients | Outpatients | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Isolate coded name | Specimen | Mer | Ceft | Cef | Enzyme produced | Isolate name coded | Specimen | Mer | Ceft | Cef | Enzyme produced |
5660 | Throat | <1 | 4 | 8 | - | 2609 | Ear | <1 | <2 | <2 | - |
5661 | Throat | <1 | <2 | <2 | - | 4278 | Ear | <1 | <2 | <2 | - |
1183 | Urine | 4 | - | 5518 | Ear | MBL | |||||
2638 | Urine | CRP | 7546 | Ear | <1 | <2 | <2 | - | |||
2844 | Urine | <1 | <2 | - | 294 | Sputum | <1 | <2 | <2 | - | |
3853 | Urine | 4 | 4 | - | 2124 | Sputum | 2 | - | |||
611 | Wound | <1 | <2 | <2 | - | 2941 | Sputum | <1 | <2 | <2 | - |
821 | Wound | 4 | 2 | - | 2966 | Sputum | <1 | 2 | 4 | - | |
971 | Wound | <1 | - | 3496 | Sputum | <1 | - | ||||
1087 | Wound | CRP | 3919 | Sputum | <1 | 4 | 4 | - | |||
1416 | Wound | <1 | 4 | 4 | - | 8142 | Sputum | <1 | 4 | 4 | - |
2064 | Wound | <1 | <2 | - | 9921 | Sputum | 2 | - | |||
2073 | Wound | CRP | 11838 | Sputum | <1 | <2 | <2 | - | |||
2581 | Wound | <1 | 8 | MBL | 1863 | Throat | <1 | <2 | <2 | - | |
2645 | Wound | CRP | 2005 | Throat | <1 | 2 | 2 | - | |||
2900 | Wound | CRP | 2047 | Throat | <1 | <2 | <2 | - | |||
3122 | Wound | <1 | 8 | 8 | - | 3238 | Throat | <1 | 4 | 4 | - |
3322 | Wound | 2 | 2 | MBL | 3477 | Throat | <1 | 2 | 2 | - | |
3451 | Wound | <1 | 8 | - | 3540 | Throat | <1 | 4 | 2 | - | |
3563 | Wound | <1 | 4 | 4 | - | 3864 | Throat | <1 | 2 | 2 | - |
3595 | Wound | <1 | 4 | 4 | - | 4087 | Throat | <1 | 4 | 4 | - |
3658 | Wound | <1 | 4 | 2 | - | 4646 | Throat | <1 | 2 | 2 | - |
3714 | Wound | MBL | 9642 | Throat | <1 | <2 | <2 | - | |||
3883 | Wound | 2 | <2 | <2 | - | 10412 | Throat | <1 | 4 | 4 | - |
4071 | Wound | <1 | 4 | 4 | - | 2383 | Tongue | <1 | 4 | 4 | - |
4082 | Wound | <1 | 4 | 4 | - | 2967 | Tongue | <1 | - | ||
4211 | Wound | <1 | 8 | - | 1408 | Urine | <1 | <2 | <2 | - | |
4212 | Wound | <1 | 4 | 4 | - | 1790 | Urine | <1 | 4 | 4 | - |
4312 | Wound | <1 | 2 | - | 2285 | Urine | <1 | 2 | 2 | - | |
4314 | Wound | <1 | 2 | 2 | - | 2588 | Urine | 2 | 4 | - | |
4354 | Wound | <1 | 2 | 2 | - | 2597 | Urine | <1 | 4 | ESBL | |
4473 | Wound | <1 | 8 | 8 | - | 2711 | Urine | <1 | 4 | MBL | |
4541 | Wound | <1 | <2 | <2 | - | 2875 | Urine | <1 | 2 | 2 | - |
5348 | Wound | <1 | 2 | 4 | - | 3199 | Urine | <1 | 4 | 4 | - |
5774 | Wound | 4 | 4 | - | 3214 | Urine | 1 | - | |||
5797 | Wound | 2 | 2 | - | 3777 | Urine | 4 | CRP | |||
6412 | Wound | <1 | 2 | 2 | - | 4188 | Urine | 4 | CRP | ||
6982 | Wound | <1 | <2 | <2 | - | 4362 | Urine | <1 | - | ||
7543 | Wound | 1 | ESBL | 5586 | Urine | <1 | <2 | <2 | - | ||
7698 | Wound | <1 | 4 | 4 | - | 8599 | Urine | 8 | - | ||
7881 | Wound | <1 | 2 | 2 | - | 10019 | Urine | <1 | 8 | - | |
8283 | Wound | <1 | <2 | <2 | - | 10600 | Urine | <1 | 4 | - | |
8702 | Wound | <1 | <2 | <2 | - | 2671 | Vag. swab | <1 | 2 | 2 | - |
8913 | Wound | <1 | <2 | <2 | - | 2689 | Vag. swab | <1 | 8 | - | |
10195 | Wound | <1 | - | ||||||||
10336 | Wound | <1 | <2 | <2 | - | ||||||
10708 | Wound | <1 | 4 | 2 | - | ||||||
10800 | Wound | <1 | 8 | CRP | |||||||
11947 | Wound | <1 | 2 | 2 | - | ||||||
19677 | Wound | <1 | 4 | 4 | - |
Mer – meropenem (S<2, I4-8, R>8); Ceft – ceftazidime (S<8; R>8); Cef – cefepime (S<8; R>8); CRP – carbapenemase-producnig
Inpatient isolates generally showed much stronger resistance than outpatient, which is in line with earlier reports (35). Similar was also the prevalence of aminoglycoside-resistant
Table 4 shows that 29 (18 inpatient and 11 outpatient) of our 94 isolates were resistant to up to three or more antibiotic classes. Fourteen originated from wounds (48.3 %) and 12 from urine (41.4 %), while only a few came from sputum and tongue. Twenty-four MDR isolates (82.8 %) produced both pyoverdine and pyocyanin (no other pigments were observed), all were non-typeable, while the P6 and P11 serotypes were equally distributed among all MDR isolates. Nine of the MDR isolates – mostly inpatient wound – were moderate biofilm producers, while 12 showed weak biofilm formation. Only one from inpatient urine formed a strong biofilm. As many as 13 MDR isolates (44.8 %) produced antibiotic-metabolising enzymes, while only two were in the group of isolates resistant to up to two antibiotic classes (Tables 3 and 4). Among these, only five (35.7 %) had the P6 serotype and produced moderate or weak biofilm and both pigments. Six isolates from the same group produced only pyoverdine and weak biofilm or none at all. In contrast, most isolates resistant to one antibiotic class were non-typeable (six isolates, 42.9 %) and had the P11 serotype (four isolates, 28.6 %). Most were moderate or strong biofilm- and pigment producers (Table 4). Thirty-seven isolates (39.4 %) were completely susceptible to all tested antibiotic classes. P1 and P6 were the most common monovalent serotypes among them (27.7 % and 24.3 %, respectively), with weak or non-biofilm formation, while the production of both pigments was again predominant.
Multidrug resistant
Pyov – Pyoverdine; Pyoc – Pyocyanin; NT – non-tippable; No biofilm producer (category -); Weak biofilm producer (category +); Moderate biofilm producer (category ++); Strong biofilm producer (category +++); Out – outpatient; In – inpatient;
In general, no direct correlation was observed between antibiotic susceptibility and virulence-associated phenotypic characteristics, serotype distribution in particular. In studies described earlier, P6 and P11 were the most abundant serogroups in all types of
Based on the results of pigmentation, serotyping, biofilm formation, enzyme production, and antibiotic resistance/susceptibility, a total of 45 isolates were selected {Table 4 [Group I – resistant to up to four antibiotic classes or more (groups 1-1 to 1-10); Group II – resistant to up to three antibiotic classes (groups 2-1 to 2-10); Group III – resistant to up to two antibiotic classes (groups 3-1 to 3-10); Group IV – resistant to one antibiotic class (groups 4-1 to 4-10); and Group V – susceptible to all tested antibiotic classes (groups 5-1 to 5-5)]} for further rep-PCR and RAPD PCR molecular genetic analysis (Table 5) to see it genetic clusterisation of different isolates in relation to phenotypic characteristics.
Selected isolates for further molecular genetic analysis
Group I | Group II | Group III | Group IV | Group V | |||||
---|---|---|---|---|---|---|---|---|---|
Resistant up to 4 antibiotic classes or more | Resistant up to 3 antibiotic classes | Resistant up to 2 antibiotic classes | Resistant up to 1 antibiotic class | Susceptible to all tested antibiotic classes | |||||
Isolate coded name | Group cipher | Isolate coded name | Group cipher | Isolate coded name | Group cipher | Isolate coded name | Group cipher | Isolate coded name | Group cipher |
2711 | 1-1 | 5797 | 2-1 | 8283 | 3-1 | 2609 | 4-1 | 3864 | 5-1 |
7543 | 1-2 | 4312 | 2-2 | 5518 | 3-2 | 821 | 4-2 | 2047 | 5-2 |
2597 | 1-3 | 5348 | 2-3 | 10195 | 3-3 | 4362 | 4-3 | 6412 | 5-3 |
3714 | 1-4 | 10019 | 2-4 | 3563 | 3-4 | 1790 | 4-4 | 3238 | 5-4 |
2645 | 1-5 | 3658 | 2-5 | 3122 | 3-5 | 7881 | 4-5 | 11947 | 5-5 |
3777 | 1-6 | 2967 | 2-6 | 10800 | 3-6 | 4211 | 4-6 | ||
2900 | 1-7 | 2844 | 2-7 | 2064 | 3-7 | 3496 | 4-7 | ||
3451 | 1-8 | 3853 | 2-8 | 11838 | 3-8 | 8142 | 4-8 | ||
2124 | 1-9 | 2588 | 2-9 | 1408 | 3-9 | 971 | 4-9 | ||
4188 | 1-10 | 9921 | 2-10 | 2383 | 3-10 | 2689 | 4-10 |
Primers 272 and 208, which were used for the RAPD PCR, created 3 to 15 fingerprinting patterns, whose range differed from 100 bp to 5000 bp. These results show that different primers within the cluster with the largest number of isolates yield similar grouping (Figure 2). Primer 272 allocated 12 clusters, while 208 allocated eight clusters. Extraordinary heterogeneity was noticed with both primers within the clusters. With primer 208 the largest cluster was extremely heterogeneous and included all but the fourth group of tested isolates was excluded. With primer 272, that same cluster was divided into as many as seven clusters. For both RAPD primers used, there was a noticeable pattern uniformity among different groups of antibiotic resistance. The case in point is pattern uniformity of the first, second, fourth, and fifth group. Similar uniformity was observed for isolate patterns of the third, first, and second group. In contrast, pattern dissimilarity (based on genetic distances) was observed between the isolates from the fourth and second group, especially with primer 272. The most genetically distant and therefore most diverse patterns were obtained for the isolates from the susceptible group, in the form of separate monophyletic branch for isolates 5-1 (primer 272) and 5-4 (primer 208).
Additionally, BOX and ERIC primers used in rep-PCR analysis created between five and 17 fingerprinting patterns of different sizes, ranging from 100 bp to 5000 bp. BOX PCR allocated a total of 10 clusters, indicating a good primer-discriminatory power, while ERIC PCR provided a total of eight clusters (Figure 3). As in RAPD analysis, the BOX PCR clusters were extremely heterogeneous, except for a few homogeneous clusters. The results of the BOX PCR analysis, like in RADP profiling, showed pattern uniformity of different antibiotic resistance groups, but certain clusters were heterogeneous. For instance, BOX PCR split the largest cluster obtained with primer 272 into four separate clusters. Pattern uniformity between isolates from the same resistance group within some clusters was also verified. The similarity between BOX and 272 primers was observed in terms of pattern uniformity between isolates from the first MDR group and those from the second and fourth group, as well as between the isolates from the fourth and fifth group. Another correlation was observed in pattern dissimilarity between isolates from the fourth and the second group, while isolate 5-1 was the most distant one genetically, as in BOX PCR analysis. ERIC PCR analysis generated more homogeneous isolate clustering from different antibiotic groups (mainly the second and the third group), and allocated a total of eight clusters. Unlike other tests, ERIC PCR showed pattern uniformity between the most susceptible isolates (the fifth group) and the second, third, and fourth group. Since RAPD272 PCR analysis provided the best characterisation with 12 clusters, we selected a few representative isolates from each cluster for 16S rRNA gene identification and all were identified as
The use of 272 and 208 primers was successful in discrimination of different
We did not find a correlation between RAPD and rep-PCR patterns and antibiotic resistance/susceptibility or other phenotype characteristics (production of pigments, distribution of serotypes and biofilm formation). The uniformity of genetic patterns of
This is the first comprehensive study of genotype and phenotype characteristics of