Antibiotic resistance, ability to form biofilm and susceptibility to copper alloys of selected staphylococcal strains isolated from touch surfaces in Polish hospital wards

Despite the employment of sanitary regimes, contact transmission of the aetiological agents of hospital infections is still exceedingly common [1]. The issue of microbe transmission becomes particularly important when multidrug-resistant microorganisms, such as methicillin-resistant staphylococci (MRSA, MRCoNS), vancomycin-resistant enterococci (VRE), or non-fermentative bacilli, appear in the hospital environment [2]. The spread of methicillin-resistant S. aureus (MRSA) or S. epidermidis (MRSE) outside hospital units (hotels, buses) is also described, but it is of greater significance when the drug-resistant bacteria present themselves in hospitals [3‐6]. Methicillin-resistant staphylococcal strains are frequently resistant not only to beta-lactam antibiotics but also to the majority of antibiotics used for therapy. Rapidly increasing resistance among both staphylococci and Gram-negative bacilli is a very serious problem in hospitals as it limits effective treatment of infections, especially in patients with severe infections treated in ICUs [7, 8]. The most common vector mediating the transfer of microbes is the hands of the staff and of patients, and the reservoir is represented by surfaces most often touched (door handles, countertops, light switches and others), on which these microorganisms can survive over long periods of time – many days or even months [9]. Cleaning and disinfection of these surfaces is an action obviously applied that has significance in controlling MRSA and MRCoNS [9]. Solutions to this problem are searched for in many directions, and understanding bacterial mechanisms of resistance enables one to seek new drugs tackling this resistance, but there is also a search for new substances that could be used in disinfectants and new materials (e.g., metal alloys) for use in commercial (touch) surfaces.

The materials for the study were strains cultured from touch surfaces in 13 different units in three hospitals of various sizes and profiles located in Małopolska province (the south of Poland). Swabs for testing were collected from the following surfaces that may be a reservoir for microbes: worktop in sickroom, bedside table top, drip stand, bed frame, soap dispenser, disinfecting fluid dispenser, light switch, ventilator monitor, mobile phone, department landline phone receiver, computer keyboard, dressing (or surgical) trolley worktop, door handle, protective glove container, and tissue package. The detailed methodology and results of environmental screening are described elsewhere [10], but the most numerous isolates were coagulase-negative staphylococci (85.7%), followed by Staphylococcus aureus (2.7%), streptococci (including E. faecalis) (8.9%), Gram-negative bacilli (1.8%) and others (0.9%). In the most numerous group of coagulase-negative staphylococci, the following species were isolated: Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus warneri, Staphylococcus capitis, Staphylococcus simulans, Staphylococcus pettenkoferi, Staphylococcus caprae and coagulase-positive ones - S. aureus.

The collected strains were stored in the Department of Microbiology at −70 °C (according to the procedure of Microorganism Preservation System Protect TS/80-MX).

Microbes of the genus Staphylococcus accounted for the vast majority of the population of bacteria cultured from touch surfaces in the hospital environment. Among staphylococci, of which almost 97% were CoNS (coagulase-negative staphylococci), there were 8 different species, the most frequent of which were S. epidermidis, S. haemolyticus and S. hominis, accounting for 34%, 30% and 15% of all CNS isolates, respectively.

The staphylococcal strains that were found on touch surfaces were primarily CoNS. Fewer than 3% of S. aureus species found were coagulase-positive isolates. Staphylococcal strains on touch surfaces can come from both the normal human flora (hand skin) and the environment, especially the air. CoNS are not dangerous to people with a properly functioning immune system but, in hospitals, they may pose a threat especially for patients who are severely ill, older, immunocompromised, hospitalized in the ICU or newborns in the NICU [19, 20]. S. epidermidis , S. haemolyticus, and S. hominis are the most frequent CoNS found to be responsible for causing skin infections as well as severe invasive infections [21‐23]. Additionally, treating infections caused by CoNS can be difficult due to their drug resistance [23, 24].

In our study, among CoNS species, S. epidermidis and S. haemolyticus were predominant, which differs from studies on air also conducted in the south of Poland where S. saprophyticus and S. warneri were prevailing [25]. The reason for this difference might be the different places of isolation (touch surfaces, not air) as well as the method applied for species identification. In this work, we employed the state-of-the-art and most recommended method, that is, Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF). In other studies in which authors employed the MALDI method for environmental research, strains from the species S. haemolyticus, S. hominis [4], S. haemolyticus and S. epidermidis [26] were most common. Additionally, Mkrtchyan et al. showed that results may vary when different identification methods are applied [27].

S. aureus, which is the main cause of hospital-acquired infections, was not isolated in large numbers, which can be surprising but has been confirmed by recent studies in Poland and abroad [4, 25, 28].

Many CoNS strains were methicillin-resistant (as many as 65%), which greatly limits therapeutic options in the case of potential opportunistic infections that they cause, due to inapplicability of the entire group of beta-lactam antibiotics [29]. For the vast majority of isolates, phenotypic resistance was confirmed through the detection of the mecA gene. Three S. epidermidis strains had the mecA gene and retained sensitivity to cefoxitin – similar strains were found for the species S. aureus [30].

Surprisingly, many isolates were also resistant to erythromycin (over 2/3) and clindamycin (a 1/2). Additionally, almost 55% of isolates had simultaneous resistance to methicillin and the MLSB mechanism of resistance.

The most frequent phenotypically detected mechanism of resistance to macrolide antibiotics, lincosamides and streptogramins B was the constitutive MLSB mechanism (35.8%), which agrees with the study by Lina [31] (here, it was 36.6%) and is a little less than Castro-Alarcon’s results [32] (47%) but different from Lenart-Boroń’s findings (where it was present only in 4% of the strains and where the inducible mechanism was predominant) [25]. The phenotype of resistance to erythromycin and retained sensitivity to clindamycin (MSB) was confirmed in 17 isolates (17.9%), and for Lenart-Boroń, it was 28% [25]. The gene associated with resistance to macrolides that was most often detected in our study was the msrA gene encoding a pump responsible for active efflux of the antibiotic from the cell (29.5% of the strains); the ermB gene, one of the genes encoding a methylase, was present in only 1 strain. Castro-Alarcon had contrasting results in which 32% of strains had the ermA gene [32]. The differences between the genes detected and the phenotypic detection of mechanisms of resistance may stem from testing only several selected genes – we did not look, e.g., at different variants of the msr gene, mph genes (encoding phosphotransferase), or the flax gene (responsible for resistance to lincosamides). We also did not examine the problem of gene expression. Our aim was not only to study the mechanisms of resistance; we also wanted to select strains for research on antibacterial properties of copper alloys.

One of the significant virulence factors of CoNS strains is their ability to form biofilm. The biofilm testing method applied here is a screening method. However, it allowed us to estimate that approximately 1/3 of the strains are potentially biofilm-forming, fewer than in the study by Szczuka, where 64% of CoNS strains formed biofilm [33].

The copper alloys that we tested have Environmental Protection Agency (USA) certificates, but EPA recommendations are based on tests for such bacterial species as Staphylococcus aureus, Escherichia coli, Enterobacter aerogenes and Pseudomonas aeruginosa, which are the bacteria that cause the majority of hospital-acquired infections [34, 35]. Coagulase-negative staphylococci, however, have also been identified as aetiologic agents of infection [12, 15, 19], and their drug resistance and ability to form biofilms, demonstrated in this study and by other researchers [24, 26], justify activity assays of copper alloys for potential use on touch surfaces in hospital departments. Four strains of the most often isolated CoNS species were selected for research on their antimicrobial properties. They differed in their resistance to antibiotics and their ability to form biofilm, and our experiments also revealed differences in susceptibility to the copper alloys.

The tests conducted showed that only for Cu-ETP was similar or higher bactericidal activity found against the studied CoNS strains than against Staphylococcus aureus – the maximum bacterial suspension density reduction from the level of approx. 10⁷ CFU/mL to 0 was observed in 120 to 240 min, depending on the strain.

Tin bronze demonstrated a faster bactericidal effect against the tested SA strain. Complete reduction in suspension density was observed after 120 min, but in the case of three of the tested CoNS strains, this time was 240 or 300 min, while for the MRSH89 strain (methicillin-resistant, non-biofilm-forming), after 300 min, the reduction in suspension density was lower by 2 log.

A similar course and degree of reduction in initial suspension density was observed for all of the examined CoNS and SA on brass CuZn37 – the boundary criterion for determining bacteriostatic effects. On CuNi18Zn20, two of the tested CoNS strains demonstrated greater susceptibility than SA.

Noyce et al. examined sensitivity to copper and bronze for three different methicillin-resistant Staphylococcus aureus strains [36]. The authors observed similar susceptibility of the tested MRSA strains to copper (C19700) – total reduction of the initial suspension density from approx. 1E + 07 to zero in under 90 min – and for two MRSA strains on bronze (C24000), they found total reduction after approximately 4.5 h, while for one of the tested strains, the degree of reduction after 360 min did not meet the qualification criterion for bacteriostatic properties [36].

The study that we conducted shows that coagulase-negative staphylococci, most often isolated from touch surfaces in Polish hospital wards [13], are not only highly drug-resistant but also perhaps less sensitive to copper alloys than Staphylococcus aureus. However, most of the CoNS strains tested in this study demonstrated susceptibility to copper alloys, which as a result are useful as a complementary solution for traditional cleaning and disinfection in hospitals.

Additionally, in Poland, there are no official recommendations for testing the antimicrobial effectiveness of materials dedicated for touch surfaces in healthcare settings. In the case of developing such recommendations, perhaps research on antimicrobial properties of materials intended for use as touch surfaces should also include representative coagulase-negative staphylococci because, as was shown in this study, they can be a reservoir of antibiotic-resistance genes. The advantage of copper as a material with antimicrobial properties intended for touch surfaces is its cidal activity, consisting of the destruction of genetic material as shown in other studies [37], and hence the possibility that it can eliminate genes for antibiotic resistance.