Gram-positive bacteria are the principal causative agents of healthcare-associated infections (HAIs) in both adults and children.Reference Wisplinghoff, Seifert, Tallent, Bischoff, Wenzel and Edmond1–Reference Blackburn, Henderson, Minaji, Meuller-Pedobdy, Johnson and Sharland3 Chlorhexidine gluconate (CHG)-based antiseptics are commonly employed in efforts to diminish the frequency of HAIs.Reference Bleasdale, Trick, Gonzalez, Lyles, Hayden and Weinstein4–Reference Toltzis, O’Riordan and Cunningham8 These strategies have been endorsed in guidelines produced by the Centers for Disease Control and Prevention (CDC) and the Infectious Diseases Society of America (IDSA) for the prevention of HAIs.Reference Mangram, Horan, Pearson, Silver and Jarvis9–Reference Yokoe, Anderson and Berenholtz11
A number of efflux pump genes in staphylococci are associated with higher minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) to CHG and other antiseptics (eg, benzalkonium chloride and cetrimide).Reference Rouch, Cram, DiBerardino, Littlejohn and Skurray12–Reference Littlejohn, DiBerardino, Messerotti, Spiers and Skurray15 In S. aureus, the plasmid-borne smr and qacA/B gene complexes have been most commonly implicated, with such organisms often being termed antiseptic tolerant (AT).Reference Smith, Gemmell and Hunter16 The incidence of AT S. aureus has increased following widespread use of CHG in hospital units, and these organisms are also associated with invasive infections.Reference McNeil, Hulten, Kaplan, Mahoney and Mason17–Reference McNeil, Kok and Vallejo20 In a recent study by our group, the presence of genotypic AT in S. aureus was independently associated with chronic medical comorbidities in the host and nosocomial acquisition of infection.Reference McNeil, Hulten, Mason and Kaplan21 The relative prevalence of these genes among S. aureus in pediatric populations varies widely, ranging from 1% to 44.5% depending on geographic location and the type of patients studied.Reference McNeil, Kok and Vallejo20, Reference Fritz, Hogan and Camins22–Reference Reich, Boyle and Hogan24 Pediatric-specific data are of relevance given the variable use of CHG in children’s hospitals, particularly in neonatal intensive care units (NICUs), arising from concerns for systemic absorption and safety in young children.Reference Chapman, Aucott, Gilmore, Advani, Clarke and Milstone25, Reference Johnson, Bracken, Tamma, Aucott, Bearer and Milstone26 We have reported that among a random sample of S. aureus isolates at Texas Children’s Hospital (TCH), 32.8% possessed either smr or qacA/B,Reference McNeil, Hulten, Mason and Kaplan21 suggesting our center represents a high prevalence region for AT.
Other important bacterial contributors to HAIs in children include the coagulase-negative staphylococci (CoNS) and Enterococcus spp.Reference Blackburn, Henderson, Minaji, Meuller-Pedobdy, Johnson and Sharland3 CoNS and enterococci can carry smr and qacA/B and their presence has likewise been associated with elevations in antiseptic MICs.Reference Hijazi, Mukhopadhya and Abbott27–Reference Prag, Falk-Brynhildsen, Jacobsson, Hellmark, Unemo and Soderquist29 However, the current literature regarding the prevalence and clinical significance of AT in these other important gram-positive pathogens is sparse.Reference Lepainteur, Royer and Bourrel30, Reference Soma, Qin, Zhou, Adler, Berry and Zerr31 Among a general adult population in Hong Kong, 13.5% of CoNS colonizing isolates were positive for qacA/B.Reference Zhang, O’Donoghue, Ito, Hiramatsu and Boost32 The prevalence of AT genes among pediatric clinical isolates of CoNS is limited to a report from a NICU in FranceReference Lepainteur, Royer and Bourrel30 as well as 18 isolates in a study at Seattle Children’s Hospital.Reference Soma, Qin, Zhou, Adler, Berry and Zerr31 We sought to determine the frequency of carriage of smr and qacA/B among bloodstream isolates of CoNS and enterococci compared to that of S. aureus at TCH. In addition, we examined the relationship between the presence/absence of these genes and clinical factors in affected patients.
Methods
Microbiology and molecular biology
Blood culture isolates of S. aureus, CoNS, and Enterococcus spp obtained in the routine course of care from October 1, 2016, to October 1, 2017, were procured from the TCH clinical microbiology laboratory. TCH is a freestanding children’s hospital and tertiary-care referral center in Houston, Texas, with 592 licensed inpatient beds and >20,000 admissions annually. All bloodstream isolates identified by the clinical microbiology laboratory are subcultured, frozen at −80°C, and banked for at least 1 calendar year. Isolates for this study were subcultured and transferred to the Edward O Mason, Jr., Infectious Diseases Research Laboratory (IDRL) where they were assigned a strain number and additional analyses were performed. A portion of all isolates immediately underwent whole-DNA extraction using QIAcube (Qiagen, Valencia, CA). For this study, only 1 bacterial isolate per episode of bacteremia was included; no colonization isolates were used in this study. CoNS and enterococci were identified to the species level in the clinical microbiology laboratory using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS, Vitek MS, bioMerieux USA, Durham, NC). Only blood cultures obtained from patients <19 years old were included in this study.
All isolates were subjected to polymerase chain reaction (PCR) assay for the qacA/B and smr genes using previously published primers.Reference McNeil, Kok and Vallejo20 A subset of PCR products from CoNS and Enterococcus isolates performed during the first PCR run underwent sequencing, were subjected to basic local alignment search tool (BLAST) algorithms, and were compared against published gene sequences (Genebank JF817385 and JF817387) to confirm the identities of the PCR products. Laboratory personnel performing these studies were blinded to clinical data.
Antimicrobial susceptibility was performed by the TCH clinical microbiology laboratory in the routine course of care using Vitek-2 (bioMerieux USA). In total, 60 CoNS isolates were selected for CHG MIC determinations in the IDRL using the macrobroth dilution method.Reference McNeil, Kok and Vallejo20 Every sixth sequential isolate was chosen, and this process was repeated until a total of 60 isolates were selected for MIC determinations.
Corresponding medical records were reviewed for all patients with attention to underlying conditions, preceding CHG use,Reference McNeil, Hulten, Mason and Kaplan21 recent surgery or hospitalization, site of acquisition of infection, and infectious diagnosis. Investigators reviewing medical records and abstracting clinical data were blinded to the results of the molecular analyses, which were performed in tandem with medical record review. The Institutional Review Board of Baylor College of Medicine approved this study.
Infection control practices
Products containing CHG are utilized in a number of infection control practices at our institution.Reference McNeil, Kok and Vallejo20 At TCH, CHG is included in central venous line (CVL) insertion and maintenance bundles. Daily CHG baths are employed at TCH for all hospitalized patients with a CVL. All patients undergoing elective surgery at TCH are encouraged to take a CHG bath the night prior to operation, and this agent is the skin cleanser of choice in our operating rooms immediately prior to incision. Additionally, daily CHG mouthwashes are routinely prescribed at TCH for hematopoietic stem cell transplant (HSCT) recipients and those with acute myeloid leukemia (AML). Notable exceptions to these rules include patients cared for in the NICU in which procedural CHG is only used in infants who are (1) at least 28 weeks old (corrected gestational age), (2) at least 7 days old, and (3) weigh at least 1,000 g. Iodophor preparations are used for procedural disinfection in infants not meeting these criteria. CHG baths are only used in infants ≥48 weeks old (corrected gestational age). Quaternary ammonium compound antiseptics are used for the cleaning of rooms and inanimate surfaces at TCH.
Definitions
Sites of acquisition of infection were considered as follows: community-acquired, community-onset healthcare-associated (CO-HCA), and nosocomial. Community-acquired infections were those occurring in otherwise healthy children with onset of signs and symptoms of infection in the community. CO-HCA infections were considered those in which signs and symptoms of infection developed in the outpatient setting in children with underlying medical conditions,Reference McNeil, Hulten, Mason and Kaplan21, Reference Hulten, Kaplan and Gonzalez33 excluding well-controlled asthma, eczema and allergic rhinitis. This definition was used to capture patients with underlying conditions that place them at higher risk for infection who may not develop infection in the hospital per se. Previous studies in S. aureus conducted by our group and others have shown that CO-HCA infections are distinct from community-acquired and hospital-acquired infections in terms of molecular features and antimicrobial susceptibility.Reference Hulten, Kaplan and Gonzalez33, Reference Wang, Hines and van Balen34 Nosocomial infections were those in which the patient developed signs/symptoms of infection ≥ 72 hours after hospital admission.Reference Hulten, Kaplan and Lamberth35
Patients were considered to have had CHG exposure in the prior 3 months if they had documented use of any CHG preparation, surgery or CVL placement at TCH (with the exception of neonates not meeting criteria as specified above) in the preceding 3 months, or diagnosis of AML or HSCT.Reference McNeil, Hulten, Mason and Kaplan21 Patients who underwent surgery but did not have other indications for CHG use were considered to have one-time CHG use; others were considered to have had recurrent CHG use. CLABSI was defined in accordance with national guidelines,Reference Mermel, Allon and Bouza36 and endocarditis was defined using the modified Duke criteria. For purposes of this study, CoNS isolates were regarded as true infections if isolated in >1 blood culture or if considered as a true infection by the treating physician. Length of stay was defined as time in calendar days from date of positive blood culture to hospital discharge. Recurrent infection was considered culture proven recurrence of infection due to an organism of the same species within 30 days of completing treatment.
Statistical analysis
With regard to CoNS infections, only true infections were included in our analyses. Categorical variables were compared using the Fisher exact test and continuous variables were compared using the Wilcoxon rank-sum or Kruskal-Wallis test as appropriate. Two-tailed P values <.05 considered statistically significant. Clinical variables associated with the presence of antiseptic tolerance genes at the P < .10 level were included in a multivariable logistic regression analysis. All statistical analyses were performed with Stata version 15 software (Stata Corp, College Station, TX).
Results
During the study period, 404 viable isolates were obtained from 389 unique patients. Moreover, 254 patients with blood cultures positive for CoNS had complete medical records and 151 of these were considered to be contaminants. After excluding these contaminants, 103 CoNS, 119 S. aureus, and 19 Enterococcus isolates (totaling 241 unique isolates from 238 patients) were included in final analyses. The median age of studied patients was 1.3 years (interquartile range [IQR], 0.2–7.9 years), and 78.4% of patients had underlying medical conditions (Table 1). Significant differences in age, comorbidities, and diagnoses existed between patients with bacteremia due to CoNS, S. aureus, and enterococci.
Table 1. General Characteristics of the Study Population
Note. IQR, interquartile range; CLABSI, central-line–associated bloodstream infection.
a Most common underlying conditions listed, categories are not mutually exclusive.
7 different CoNS species were identified by MALDI-TOF MS: S. epidermidis (n = 70), S. hominis (n = 22), S. haemolyticus (n = 4), S. capitis (n = 3), S. warnerii (n = 2), S. lugdenensis (n = 1), and S. saprophyticus (n = 1). Most enterococci were E. faecalis (n = 16, 84.2%) with the remainder being E. faecium (n = 3). Among S. aureus isolates, 25 of 119 (21%) were methicillin resistant.
Antiseptic tolerance genes
Overall, 83 of 103 CoNS isolates (80.6%) possessed either qacA/B or smr compared to 44 of 119 of S. aureus isolates (37%) and 7 of 16 E. faecalis isolates (43.8%) and none of 3 E. faecium isolates (P < .001) (Fig. 1). The qacA/B gene was detected in 16 of 119 S. aureus isolates (13.4%), 73 of 103 CoNS isolates (70.8%), and 7 of 16 of E. faecalis isolates (43.8%; P < .001). The smr gene was detected in 28 of 119 S. aureus isolates (23.5%), 34 of 103 CoNS isolates (33%), and 4 of 16 E. faecalis isolates (25%; P = .28). Both AT genes were detected in 24 of 103 CoNS isolates (23.3%), 4 of 16 E. faecalis isolates (25%), and none of the S. aureus isolates (P < .001). The proportion of CoNS isolates with AT genes was similar among isolates considered true infections (83 of 103, 80.6%) and contaminants (128 of 151, 84.7%; P = .39). Sequenced qacA/B- and smr-PCR products obtained from CoNS and E. faecalis were identical to published sequences of these respective genes in S. aureus.
Fig. 1. Comparison of proportion of isolates with antiseptic tolerance genes in staphylococci and enterococci (n = 241). Simple proportions of isolates bearing genes of interest were compared using the Fisher exact test.
Antiseptic tolerance
In total, 60 CoNS isolates had CHG MICs, as determined by broth dilution. Isolates with both qacA/B and smr had higher MICs than those without these genes (MIC90, 0.25 vs 0.03 µg/mL; P = .06) (Supplemental Table 1).
Antiseptic tolerance and clinical variables
Among patients with CoNS infections, a greater proportion of qacA/B-positive strains were associated with nosocomial infections than qacA/B-negative isolates (41 of 73 [56.1%] vs 5 of 30 [16.7%]; P < .001) (Table 2). The smr-positive CoNS infections were more often associated with nosocomial acquisition (21 of 34 [61.8%] vs 25 of 69 [36.2%] P = .02) and a longer median length of hospital stay (14.5 vs 6.5 days; P = .02) (Table 3). When considered as a group, antiseptic tolerant CoNS were associated with a higher rate of nosocomial acquisition of infection (46 of 83 [55.4%] vs 0 of 20; P < .001) (Supplemental Table 2). We observed no differences in any other examined clinical variable between antiseptic-tolerant and -susceptible CoNS.
Table 2. Comparison of Patients with Bacteremia Due to qacA/B-Positive Versus qacA/B-Negative CoNS
Note. IQR, interquartile range; CLABSI, central-line–associated bloodstream infection; CHG, chlorhexidine gluconate; CVL, central venous line.
a No patients with CoNS bacteremia had a community-acquired infection.
Table 3. Comparison of Patients with Bacteremia Due to smr-Positive Versus smr-Negative CoNS
Note. IQR, interquartile range; CLABSI, central-line–associated bloodstream infection; CHG, chlorhexidine gluconate; CVL, central venous line.
a No patients with CoNS bacteremia had a community-acquired infection.
Antiseptic-tolerant and -susceptible S. aureus were also compared (Table 4). Patients with AT S. aureus infections were younger, had more medical comorbidities, more recent hospitalizations, and more exposure to CHG and antibiotics. They more often acquired infection in the hospital and more often experienced infection recurrence. Additionally, AT S. aureus isolates were more often associated with a diagnosis of CLABSI (15 of 44 [34.1%] vs 13 of 75 [17.3%]; P = .046) and less often with the diagnosis of musculoskeletal infection (2 of 44 [4.5%] vs 34 of 75 [45.3%]; P < .001) than antiseptic susceptible infections. In multivariable analyses, nosocomial/CO-HCA infections were more often associated with AT in S. aureus, whereas musculoskeletal infections were less often associated with AT. Among enterococci, there was no significant association with AT genes and any examined clinical variable.
Table 4. Comparison of Patients With Bacteremia Due to Antiseptic-Tolerant Versus -Susceptible S. aureus
Note. OR, odds ratio; CI, confidence interval; IQR, interquartile range; CLABSI, central-line–associated bloodstream infection; CHG, chlorhexidine gluconate; CVL, central venous line.
a In multivariable analyses, age was dichotomized as > vs ≤ 0.33 years (the bottom quartile of age for all patients with S. aureus bacteremia).
b Acquisition of infection is dichotomized as healthcare associated (CO-HCA and nosocomial) vs community-acquired.
c 3 patients with antiseptic-tolerant S. aureus and infection recurrence had CLABSI initially treated with CVL in situ compared to 1 patient in the antiseptic-susceptible group with infection recurrence. If these 4 patients are removed from the analyses, a statistically significant relationship between antiseptic tolerance and infection recurrence remains (5/41 [12.2%] vs 1/74 [1.4%]; P = .03).
Discussion
Despite advances in infection control and prevention, HAIs still cause substantial morbidity in both adults and children, and S. aureus, CoNS, and enterococci are significant contributors.Reference Blackburn, Henderson, Minaji, Meuller-Pedobdy, Johnson and Sharland3 Antiseptics have been demonstrated to be an effective means to reduce the incidence of HAIs; however, with widespread use of these agents, concerns have been raised in recent years regarding the emergence of reduced susceptibility.
We investigated the prevalence of AT genes among staphylococci and enterococci causing bacteremia at a large children’s hospital. Previous studies in CoNS have reported that 13%–80% of isolates possessed AT genes.Reference Hijazi, Mukhopadhya and Abbott27, Reference Lepainteur, Royer and Bourrel30–Reference Zhang, O’Donoghue, Ito, Hiramatsu and Boost32 We found that 80.6% of CoNS isolates possessed either qacA/B or smr. The proportion of isolates with AT in our study is higher than that reported at most other institutions, particularly those centers caring for children. Soma et alReference Soma, Qin, Zhou, Adler, Berry and Zerr31 reported that 65% of 18 CoNS isolates recovered from skin swabs studied at Seattle Children’s Hospital bore qacA/B. A similar proportion of CoNS were positive for qacA/B among a study of CLABSI isolates obtained from very-low-birth-weight infants in France.Reference Lepainteur, Royer and Bourrel30 The high frequency of qacA/B-positive CoNS in our population is comparable to a study from the United Kingdom in which 80% of bloodstream S. epidermidis isolates carried qacA/B.Reference Hijazi, Mukhopadhya and Abbott27 In our center, the frequency of detection of AT genes in CoNS was much higher than that detected among S. aureus (36.2%). Notably, however, the proportion of S. aureus isolates positive for AT genes was higher than that reported by other studies in pediatric populations (1%–18.5%)Reference Fritz, Hogan and Camins22–Reference Reich, Boyle and Hogan24 but was consistent with prior research at our institution.Reference McNeil, Hulten, Mason and Kaplan21, Reference Fritz, Hogan and Camins22
In previous studies of S. aureus conducted at our institution, the presence of AT genes was independently associated with nosocomial infections and underlying medical conditions in the host.Reference McNeil, Hulten, Mason and Kaplan21 In the present study, AT S. aureus were again associated with underlying medical conditions and nosocomial acquisition of infection as well as a number of other clinical factors in univariable analyses including recent hospitalization and the diagnosis of CLA-BSI. Importantly, we noted a statistically significant association with the presence of genotypic AT in CoNS and nosocomial acquisition of infection. These findings suggest that the healthcare environment and/or medical complexity select for AT in other organisms in addition to S. aureus. This is of particular importance given that previous studies examining the epidemiology and clinical impact of AT have largely focused on S. aureus with little attention given to other pathogens. CoNS was the predominant cause of all pediatric HAIs in a multinational study,Reference Zingg, Hopkins and Gayet-Ageron37 highlighting the clinical significance of this pathogen. We found no other clinical variables to be associated with AT in CoNS; the small number of healthy patients with true CoNS infections likely limited the degree to which other clinical factors could be associated with AT in these organisms. In contrast to our findings in staphylococci, we observed no relationship between AT genes in enterococci and any examined clinical variable; this was likely a consequence of the small number of enterococci studied.
We have previously reported an association with AT in S. aureus and invasive infections as well as prolonged hospital stay.Reference McNeil, Hulten, Mason and Kaplan21 In the present study, we found an association with genotypic AT in S. aureus and recurrence of infection; however, the reasons for this observation are unclear. Studies using ex vivo and in vivo models suggest that S. aureus expressing such multidrug-resistance efflux pumps may have an enhanced ability to colonize surfaces as well as cause disease.Reference Truong-Bolduc, Villet, Estabrooks and Hooper38, Reference Ding, Onodera, Lee and Hooper39 Possibly, if such organisms have an enhanced colonization capacity, they may be more likely to cause recurrent infection despite appropriate treatment. This finding is, in part, also likely related to the higher rate of CLA-BSI in the tolerant group and recurrences occurring as a result of not removing infected central lines in a minority of cases. Also, this phenomenon might be a consequence of virulent strain types that happen to possess AT genes rather than a consequence of the genes themselves. Such findings further highlight the potential impact of AT strains for public health. Notably, however, the finding of higher recurrence in AT S. aureus infections may have been a result of these patients having a greater degree of medical complexity and thus being more likely to fail treatment.
Additional limitations to this study should be acknowledged. Foremost, these findings are from a single center with a previously described high prevalence of AT S. aureus Reference McNeil, Kok and Vallejo20, Reference McNeil, Hulten, Mason and Kaplan21; thus, our findings may not be generalizable to all regions. The retrospective nature of the study limits the degree to which clinical risk factors can be definitively associated with AT organisms. Additionally, given that colonization cultures were not examined in this study, we are unable to assess the impact of AT organism colonization on risk of subsequent bacteremia or the potential mitigating effects of antibiotic/antiseptic use.
In conclusion, genotypic AT is common among bloodstream staphylococci and E. faecalis isolates at our institution. The presence of AT genes was strongly associated with nosocomial acquisition of infection in staphylococci, implying a role for the hospital environment in selecting for these pathogens. Larger studies are needed to further explore and validate these findings.
Supplementary material
To view supplementary material for this article, please visit https://doi.org/10.1017/ice.2019.3.
Author ORCIDs
J. Chase McNeil, 0000-0002-0232-7491
Acknowledgements
We are grateful to Ms Linda Lamberth as well as the staff of the TCH clinical microbiology laboratory for their assistance with this project.
Financial support
This study was funded by the National Institute of Allergy and Infectious Diseases (NIAID grant no. K23AI099159) and The Texas Children’s Hospital Pediatric Pilot Research Fund (both to J.C.M.).
Conflicts of interest
Dr Kaplan serves as the primary investigator (PI) on an investigator-initiated clinical trial sponsored by Allergan as well as the site PI of a clinical trial sponsored by Merck. Dr McNeil serves as a co-investigator on these studies, neither of which is directly related to the present work. No authors have significant financial conflicts of interest.
