Oral colonisation by antimicrobial-resistant Gram-negative bacteria among long-term care facility residents: prevalence, risk factors, and molecular epidemiology

Extensive consumption of antibiotics has been the crucial pressure impelling drug-resistance, leading to the global concern of multi-antimicrobial resistant microorganisms. While much attention has been paid to infectious diseases by multidrug-resistant (MDR) organisms in severely ill patients in intensive care units, the colonisation by antimicrobial-resistant bacteria (ARB) among patients in long-term care facilities (LTCFs) has been thus far neglected. Since the early 1970s, various reports have repeatedly emphasised the role of asymptomatic patients in LTCFs as a reservoir of ARB thus favouring their persistent colonisation and extensive dissemination throughout hospital settings [1].

A similar situation is developing across Japan, where the percentage of elderly over 65 years is predicted to be as much as 38.1% [2] by 2060, whilst health care-associated infection management in LTCFs is straggling in comparison with the one in medical settings. Adequate active surveillances still have to clarify the colonisation rates by MDR bacteria among the elderly residing in LTCFs nationwide [3]. Moreover, surveillances for ARB carriage among LTCFs residents have been almost exclusively for faecal or rectal samples [4].

The oral cavity is constantly exposed to external material during its function of air, beverage, and food intake, thus frequently exposed to various microorganisms. Specific histological features of the mouth (e.g., the papillary dorsal tongue, the mucosal epithelia of cheeks, the teeth enamel, and the periodontal surfaces), in addition to changes in the ecological condition (e.g., the salivary flow) may result in the accumulation of host-produced extracellular pellicles and the subsequent formation of a biofilm, a favourable matrix for nutrition supply. Exposure of bacteria to such pellicle-facilitated surfaces over several hours promotes the colonisation by those pathogens in this biotic niche [5]. The persistence of such organisms in the oropharyngeal area, in turn, places the patients at risk for bacterial pneumonia [6].

While previous published data of this target population mainly focused on faecal or rectal samples to evaluate the carriage of ARB [4], a survey by March A. et al. indicated a proportion of ARB present in the oropharyngeal area in addition to the other sites [7]. However, to date, few researchers have addressed this issue in the oral cavity. For those reasons, this paper addresses (i) the prevalence of oral colonisation by ARB, including extended-spectrum ß-lactamases (ESBLs)-producing and carbapenem-resistant bacteria, among the LTCF residents, (ii) risk factors associated with colonisation by AMR organisms, and (iii) molecular epidemiology to figure out if any further necessary preventive measures should be implemented.

ARB, as defined by growth on screening agar plates, most commonly Acinetobacter, Enterobacteriaceae, and Pseudomonas spp., were detected in 37 of 98 (38%) LTCF residents. Moreover, some residents were colonised with more than one type of ARB. The presence of ARB in the oral niche poses a potential risk for pulmonary infections, in which certain kinds of Gram-negative bacteria such as P. aeruginosa, E. coli, K. pneumonia and the others have been reported [6]. Our findings reinforced the significant role of LTCF residents as reservoirs for ARB and also brings the attention to the colonisation by ARB in the oral environment besides the other well-documented body areas.

E. coli ST131 has been reported to be responsible for serious extraintestinal infections and medical implications [29]. Meanwhile, the rapid emergence and international dissemination of this clonal group has posed a critical threat to public health due to its significant AMR [30]. In our study, we also determined that all ESBL-producing E. coli isolates belonged to the most-virulent phylogroup B2, were bla_CTX-M-27-producing O25:H4 ST131 fimH30 E. coli, and carried mutations in DNA sequences of the chromosomal QRDRs resulting in fluoroquinolone resistance. The mutations in the gyrA QRDR have been found to be the most frequent mutations among fluoroquinolone-resistant E.coli isolates [31]. The high virulence and resistance of fimH30 lineage to fluoroquinolones, one of the most widely used antimicrobials for urinary tract infections, has made this lineage the most epidemiologically successful subclone of E. coli ST131 [32]. Considering the severe complications, MDR, and rapid propagation of this endemic clone, infection control and transmission prevention should be earnestly considered, especially within the vulnerable population in long-term care settings.

Acinetobacter has been recently responsible for multihospital outbreaks in temperate countries increasing the risk for residents in LTCFs, especially those receiving mechanical ventilation [33]. In our study there was no MDR Acinetobacter but seven out of 10 showed susceptibility to antimicrobials according to the MIC determination, six of which showed extensive biofilm production and showed a highly resistant phenotype on screening media. Besides the detection of ARB carrying multiple AMR genes, the presence of isolates with low MIC based on biochemical tests and no acquired AMR genes while still growing on CHROMagar™ ESBL or CHROMagar™ mSuperCARBA plates, and high biofilm-formation ability, was also a noteworthy finding. This inconsistence, in fact, reflects the difference in the AMR phenotype between a planktonic lifestyle of bacteria in MIC biochemical tests and biofilm lifestyle on agar plates, where bacteria are encased in an extracellular matrix that provides them tolerance and resistance mechanisms to combat antimicrobial challenges. For instance, the biofilm matrix provides a barrier for the penetration of antibiotics and hence, decreases bacteria susceptibility. Exopolysaccharides and extracellular DNA play a role in the resistance to antimicrobial agents, or secreted ß-lactamases in the matrix can degrade antimicrobials [34]. The growth of bacteria in the presence of an antimicrobial agent, despite their low MICs by Vitek-2 or the lack of AMR genes, may suggest their growability inside the human body with the presence of that antimicrobial agent. In other words, the low MIC of Vitek-2 does not necessarily mean the bacteria cannot grow in the presence of antimicrobials inside the human body, considering their biofilm lifestyle. From the viewpoint of hospital infection control, the mobile AMR genes or plasmids are significant concerns given their horizontal gene transfer from organism to organism within the health-care setting. However, from the viewpoint of clinical implications, both the AMR gene-carriers and biofilm-producers are important for their resistance phenotype inside the human body, either through the enzyme-mediated mechanism or the biofilm-based mechanism or through a combination of both. Moreover, our six isolates appeared to be clonally disseminated among the hospital ward. Peleg et al. emphasised that the attachment and biofilm formation ability of A. baumannii inside the body and on object surfaces took part in their virulence mechanisms and long endurance within health care settings, thus increased the potential for an epidemic outbreak [35]. Our results also support this hypothesis and lead to the conclusion that strong biofilm-producing A. baumannii should be regarded as a potential risk even though it lacks AMR genes.

A. ursingii 56C showed expanded resistance to various ß-lactams. A. ursingii is an uncommon opportunistic pathogen first described as a novel species in 2001 [36]. Sporadic cases involving serious bloodstream infections, in patients that are either immunocompromised or immunocompetent, have been reported [37, 38]. Although most of these isolates were susceptible to antimicrobial agents, the first report of a carbapenem-resistant A. ursingii from clinical isolates was described in Japan in 2010 [39] followed by a report of three carbapenemase-producing A. ursingii isolates in The Netherlands from September 2015 to June 2016 [40]. In our study, the A. ursingii isolate did not carry any carbapenemase gene such as bla_IMP-4 and bla_OXA58 but had some AMR genes as the ones carried by the isolates from The Netherland, for example, bla_CARB-2, aac(6′)Ib-cr, mph(E), msr(E), sul1, and tet(39).

Another leading emerging nosocomial pathogen that challenges the therapeutic treatment in clinical settings is P. aeruginosa, which confers both AMR phenotype and robust biofilm production, the so-called resistant biofilm-phenotype. Alternatively, Leibovitz et al. designated the biofilm covering the nasogastric-feeding tubes as reservoirs for biofilm-associated persistent P. aeruginosa in the oropharynges [41]. MBL genes coexist with other AMR elements and are commonly carried on mobile gene cassettes in class 1 or 3 integrons inserted into plasmids or in the chromosome [42], thus MBL-producing P. aeruginosa generally has a MDR phenotype. A 2005 outbreak of MDR P. aeruginosa carrying a novel chromosomally encoded class 1 integron, namely In113, later designated as type E integron by our group [8], has been reported in a neurosurgery ward in the Miyagi prefecture [9]. Complete genome sequence of a representative of this endemic cluster, NCGM2.S1 (previously named IMCJ2.S1) revealed that this strain harboured a bla_IMP-1 gene cassette and a aac(6′)-Iae gene cassette in the integron In113, promoting the high level of MDR to ß-lactams and aminoglycosides, respectively, without the presence of any plasmids [10]. In the meantime, an epidemic clonal (designated as type F by our group) MDR P. aeruginosa in the Hiroshima region, represented by PA058447, was also noticed from a 9-year longitudinal molecular epidemiology study. This strain was proposed to have identical resistance element sequences as In113-carrying P. aeruginosa (type E) yet a disrupted Intl1 by an IS26 insertion and subsequent genomic reorganisation [11]. Interestingly, those two endemic clones were identified as having the same sequence type, ST235. Our detection of ST235 P. aeruginosa 71E with outstanding biofilm-formation ability, MDR phenotype, and close clonal origin with the epidemic ST235 MDR P. aeruginosa in Hiroshima region has called a conscientious attention.

Although S. maltophilia was not deeply investigated due to its natural resistance, the high prevalence of this species in this LTCF was also noteworthy since this pathogen has been recognised to produce nosocomial pneumonia in patients that are critically ill [43]. Inadequate empirical antimicrobial therapy over such high intrinsic resistance species may fail to defeat the infection, thus caution should be taken when administering medication empirically to those individuals.

Strokes, predominantly cerebral infarction and cerebral haemorrhage, were found to be a significant risk factor for oropharyngeal colonisation by ARB in LTCF. Gill et al. previously stated that stroke was significantly associated with disability in the elderly [44], requiring substantial assistance from care-givers with regards to dressing, toileting, eating, and other daily activities. In addition, faecal incontinence associated with diaper use and nursing aids have been recognised as risk factors for faecal carriage of ESBL-Enterobacteriaceae [45] and were likely linked to ARB transmission mediated via healthcare workers. This explanation is plausible as 14.5% of LTCF staff were colonised with ESBL-producers in the study of March et al. [7]. Besides, contaminated environments and mechanical equipment (e.g., nasogastric tubes, tracheostomy tubes, and urinary catheters) may accelerate the colonisation by nosocomial pathogens specifically those showing vigorous biofilm production. An outbreak of AMR A. baumannii due to the utilisation of contaminated tap water from hygiene sinks for oral care in an intensive care unit was reported [46]. The greater the advanced disability patients have, the more assistance and interaction with care-workers and medical-device operators are required, consequently facilitating the propagation of such microorganisms. In addition, patients who were fed with PEG tubes might undergo a reduction in mastication activity and salivary secretion, hence promoting the pathogenic colonisation inside the oral cavity. Since colonisation is an essential prerequisite for infection [45], the colonisation by ARB in LTCF raises a crucial challenge regarding the shortage of available efficient anti-infective agents once infection occurs.

Recently, to reduce the risk of “nursing home-acquired pneumonia” (NHAP), more effort have been taken to improve oral care such as implementing professional mouth care (brushing teeth, swabbing the mucosa, cleaning dentures, using mouthwash, having dental check-ups by professional dentists, or a combination of those methods) [47], being aware of dysphagia signs [48], improving the staff practices for oral care [49], or promoting oral hygiene, and swallowing ability of the LTCF residents [50]. Professional oral health care was proved to be cost-effective in declining the mortality and morbidity rates from NHAP in German healthcare settings [51]. In a systematic review by the Cochrane Library, despite the lack of high-quality evidence to determine the most effective measures to prevent NHAP, these studies proposed the efficacy in lowering pneumonia mortality through professional oral care when compared to usual oral care after a 24 month-follow up [47]. Whether the professional oral care will be an effective measure to reduce prevalence of ARB remains to be examined.

The study has several potential limitations that deserved to be discussed. First, as a limited number of patients participated in this surveillance, alternative possible risk factors might have not been identified. Second, no historical records of antibiotic use among the residents one year prior to this study were available, hindering the determination of the association between antimicrobial therapy and development of ARB. Third, this surveillance was conducted in a single LTCF, thus the overall prevalence of oral colonisation by ARB among other LTCFs in Japan remains to be elucidated. A similar study should be conducted in a large-scale long-term care system in order to comprehend the general situation and comparative evaluation.

In summary, the present study detected high prevalence of AMR Gram-negative bacteria, ESBL-producing and carbapenem-resistant pathogens relevant to aspiration pneumonia, which carried the resistance genes on mobile elements such as plasmids or integrons or in the chromosome and/or are strong biofilm producers, in the oral cavity of LTCF residents. Health care workers involved in oral care should be aware of such ARB and pay special attention to transmission prevention and infection control to diminish the dissemination of ARB or the mobile resistance elements in LTCFs. Last but not least, with the rapid ageing of the Japanese society, surveillance initiatives and regional and national projects for infection control should not put aside the significant role of LTCFs or nursing homes in the healthcare network.