Bacterial sensitivity to chlorhexidine and povidone-iodine antiseptics over time: a systematic review and meta-analysis of human-derived data

Surgical site infection (SSI) is the most common complication of surgery, increasing healthcare costs and hospital stay. Chlorhexidine (CHX) and povidone-iodine (PVI) are used for skin antisepsis, minimising SSIs. There is concern that resistance to topical biocides may be emergeing, although the potential clinical implications remain unclear. The objective of this systematic review was to determine whether the minimum bactericidal concentration (MBC) of topical preparations of CHX or PVI have changed over time, in microbes relevant to SSI. We included studies reporting the MBC of laboratory and clinical isolates of common microbes to CHX and PVI. We excluded studies using non-human samples and antimicrobial solvents or mixtures with other active substances. MBC was pooled in random effects meta-analyses and the change in MBC over time was explored using meta-regression. Seventy-nine studies were included, analysing 6218 microbes over 45 years. Most studies investigated CHX (93%), with insufficient data for meta-analysis of PVI. There was no change in the MBC of CHX to Staphylococci or Streptococci over time. Overall, we find no evidence of reduced susceptibility of common SSI-causing microbes to CHX over time. This provides reassurance and confidence in the worldwide guidance that CHX should remain the first-choice agent for surgical skin antisepsis.

www.nature.com/scientificreports/ oxidising proteins, nucleotides and fatty acids 17 causing cell death. Both PVI and CHX are active against gram positive and negative bacteria, fungi and viruses 16,18 .
There are growing fears that as antiseptic use increases, sensitivity may reduce and resistance emerge 19 . This is particularly concerning given the accelerating global antibiotic resistance crisis 20 . Multiple bacteria have shown reduced sensitivity (perhaps even resistance) to CHX, particularly through the multidrug resistance efflux protein qacA 19 . Methicillin resistant Staphylococcus aureus (MRSA) samples with qacA/B genes showed persistent MRSA carriage despite de-colonisation therapy 21 . Not only is the presence of reduced sensitivity concerning, but the presence of resistance conferring genes seems to be increasing annually 22 . PVI resistance has been less commonly reported 23 although this might be secondary to the multimodal effect of iodine on microbes 23 or otherwise. Overall, the current state of microbial sensitivity to CHX and PVI remains unclear.
The aim of this review is to summarise the sensitivity profiles of skin microbes (relevant to surgical site infection) to CHX and PVI, and explore how these have changed over time.

Methods
This review was designed and conducted in accordance with the Cochrane Handbook of Systematic Reviews 24 , the protocol was published in the PROSPERO databased (CRD42021241089) and the report has been authored in accordance with the PRISMA checklist 25 .

Types of studies.
We included all studies which reported the resistance of microbes to CHX or PVI based topical biocides derived from human samples. There were no language restrictions. We excluded case reports and studies which used antimicrobial solvents (e.g., alcohol) or mixtures of antiseptics (e.g. chlorhexidine mixed with cetrimide).
Search strategy. The NICE Healthcare Databases (hdas.nice.org.uk) was searched according to Appendix 1 (Supplementary Materials). The medRxiv and bioRxiv preprint archives were searched with the same strategy using the R package medrxivr 26 . This yielded 582 hits in PubMed, 720 in Embase, 993 in Web of Science and 896 in CINAHL. After de-duplication, there were 2318 unique citations which were screened (Fig. 1). A further 3 articles were found by manual searching of these articles. Outcomes. The outcome of interest was the minimum bactericidal concentration (MBC). MBC was chosen over the minimum inhibitory concentration (MIC) as a measure of susceptibility because it is generally felt to be a more appropriate for topical antiseptics 15,28 . We were interested in deriving a pooled estimate of the MBC for different species to understand how this may have changed over time.
Methodological quality assessment. The risk of bias was not assessed because there are no validated tools available for studies of this nature and the study selection process ensured that only the highest quality research was included.
Missing data. After back-calculating some parameters, the overall rate of missing data (in the predictor variables) was 14.5%. Importantly, the variance of the mean MBC was missing at random in 36 observations (61%) and because this was required for the primary analyses, we imputed this data using chained equations 29,30 . Statistical analysis. The raw data are available open-source at https:// osf. io/ khnb2. Using metafor 31,32 , 5 studies 33-37 reporting the MBC of CHX for Staphylococci were identified as outliers (based on externally standardised residuals) or influential studies (based on the Cook's distance and leave-one-out values of the test statistics for heterogeneity), and their 95% confidence intervals (CIs) were far outside the 95% CI of the pooled estimate, so they were excluded from the meta-analyses. Data were then analysed in Stata/MP v16 (StataCop LLC, Texas) using the meta suite. To synthesise a pooled MBC, we used a mixed-effects meta-analyses. We sub-grouped by the microbial family. To estimate change in MBC over time, we performed meta-regression for Staphylococci and Streptococci, separately. A sensitivity multivariable meta-regression for Staphylococci was performed, controlling for methicillin-resistance as a binary co-variate. The REML estimator was used throughout. To align with calls for the abolition of p-values, we minimise their use and avoid the term "statistical significance" 38,39 , instead focusing on how our findings may be clinically applicable and what might explain uncertainty in the estimates.

Study characteristics. The details of the included studies are summarised in
Evidence synthesis. The MBC of CHX differed significantly between the families of microbes (Fig. 2) Fig. 4).
There were insufficient data for meta-analysis of the MBC of PVI. Also, the majority of MBC data for PVI was derived from studies of Enterobacteriales, which is not a common cause of surgical site infection and so equally, not clinically relevant.

Discussion
This review summarises the evidence to-date and suggests that there has been no increase in MBC of CHX for the main SSI-causing skin microbes in recent decades. The stability of CHX susceptibility is reassuring for clinicians and policy makers alike, as it endorses current surgical guidance worldwide, which advocates topical alcoholic chlorhexidine for skin asepsis prior to surgery. The definition of bacterial susceptibility and resistance to biocides is still a matter of debate. Most clinically relevant bacteria have defined susceptibility and resistance to systemic antibacterials based upon the MIC relative to an epidemiologically derived clinical breakpoint. However, the use of MICs is less useful in determining the efficacy of topical antiseptics biocides given the desire to induce death of specific microbes rather than inhibition. Understanding the lethality of a biocide (hence MBC) is therefore a potentially more attractive measure than MIC for topical antiseptics 114 . Additionally, the relevance of both MICs and MBCs with respect to biocides has been questioned 15 . Chlorhexidine for skin prep is used in concentrations of 5000 to 50,000 µg/mL and with MBC values ranging from 0 to 30 µg/mL, this is one thousand times greater than the apparent required concentration 115 .
MIC and MBC rely on attaining a steady concentration in bodily fluids as seen by the pharmacodynamics of antibiotics 116 . The EUCAST definition of a susceptible organism is "a microorganism is defined as susceptible by www.nature.com/scientificreports/ a level of antimicrobial activity associated with a high likelihood of therapeutic success" 117 . Therapeutic success in the context of topical CHX prior to surgery is disinfection; complete elimination of all relevant micro-organisms, except certain bacterial spores 118 . In the literature, there are no established clinical breakpoints for defining resistance and susceptibility of skin flora to CHX. With the available data, we look at if there is a drift in MBC over time as the next best surrogate for the development of resistance in skin flora known to cause SSI. Measurement of MIC and MBC are gained from in vitro susceptibility testing of microorganisms to topical biocides and therefore, provide little information as to the mechanism of resistance or likely clinical outcome. Therefore, our recommendation is the utilisation of epidemiological cut-off values (ECOFF) based on MBC distributions to better understand the response of bacteria to CHX in clinical practice. An analysis of ECOFF values of CHX to common bacterium, including those commonly causing SSI did not reveal a bimodal distribution, concluding that resistance is uncommon to CHX in natural populations of clinically relevant organisms 88 . While values above a certain cut off may be defined as a breakpoint and hence resistant, this needs to be correlated with the clinical picture. Does CHX still achieve adequate disinfection in a population of bacterium with a MBC value greater than the 95% ECOFF? Without this information, our understanding of how MBC values beyond the normal distribution impacts clinical use remains poor. Of note, all the studies measuring MBC, the method of analysis looks at the action of a biocide over a very long period (hours). This does not represent real time clinical application of CHX, whereby topical application occurs over minutes although equally, chlorhexidine is known to penetrate the stratum corneum and exert bactericidal activity for hours (and potentially days) after application 73 . Therefore, we propose alternative methods of testing  www.nature.com/scientificreports/ biocides utilising a model of topical application over minutes using clinically relevant concentrations (such as those conducted by Touzel et al. 107 might be more meaningful. Furthermore, to represent clinical use, experiments must also be conducted looking at biofilm models of skin flora and the impact of CHX use on biofilms. The majority of experiments utilising CHX and skin flora carried out in-vitro measurements using pure monospecies planktonic forms of bacteria, which does not represent the clinical environment of skin flora. Additionally, if a rise in MBC is seen, this does not prove that chlorhexidine contributes to an increase in antiseptic tolerance. We propose that future in vivo studies apply CHX topically and explore how antiseptic and antibiotic tolerance or resistance develops thereafter. Until these methods are mature, cost-effective and widely available, the MBC is the best surrogate for emergent resistance.

Limitations
Most of the included studies reported MIC rather than MBC, which meant that much data could not be synthesised. This might explain why our meta-data (for Staphylococci and Streptococci at least) disagrees with individual articles 18,19,21,22 . Papers also often failed to report the type and concentration of chlorhexidine used. Some papers clearly stated the year that the microbial strain was isolated, although this was often unclear and therefore the date of the paper was taken as the year of the isolate which may not adequately represent the change in MBC over time. This was also the case with location, so where possible the location of the laboratory or hospital was used as a surrogate. Isolates from a clinical setting are exposed to different selection pressures; as 61% of the microbes used were laboratory isolates as opposed to clinical isolates, it is unclear how our data can be generalised to clinical environments.

Conclusion
There has been no demonstrable change in the susceptibility of surgical site infection causing pathogens to chlorhexidine over time. A clear definition of reduced susceptibility and resistance of pathogens to biocides is needed, alongside consensus on the methods for measuring these phenomena.

Data availability
The raw data are available via the Open Science Framework (https:// osf. io/ khnb2/). The statistical syntax are available from the senior author (RGW) upon request.