Emerging ST121/agr4 community-associated methicillin-resistant Staphylococcus aureus (MRSA) with strong adhesin and cytolytic activities: trigger for MRSA pneumonia and fatal aspiration pneumonia in an influenza-infected elderly.

The pathogenesis of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) pneumonia in influenza-infected elderly individuals has not yet been elucidated in detail. In the present study, a 92-year-old man infected with influenza developed CA-MRSA pneumonia. His CA-MRSA was an emerging type, originated in ST121/agr4 S. aureus, with diversities of Panton-Valentine leucocidin (PVL)(-)/spat5110/SCCmecV(+) versus PVL(+)/spat159((etc.))/SCCmec (-), but with common virulence potentials of strong adhesin and cytolytic activities. Resistance to erythromycin/clindamycin (inducible-type) and gentamicin was detected. Pneumonia improved with the administration of levofloxacin, but with the subsequent development of fatal aspiration pneumonia. Hence, characteristic CA-MRSA with strong adhesin and cytolytic activities triggered influenza-related sequential complications.

Introduction

Staphylococcus aureus is a major human pathogen that causes skin and soft-tissue infections, life-threatening infections such as pneumonia and sepsis, and toxinoses including toxic shock syndrome. Methicillin-susceptible S. aureus (MSSA) has evolved as methicillin-resistant S. aureus (MRSA) through the acquisition of staphylococcal cassette chromosome mec (SCCmec) [1], [2], [3].

Two types of MRSA have been identified [1]: traditional MRSA, which emerged in hospitals in 1961 and is now classified as healthcare-associated MRSA (HA-MRSA) with global examples of ST239/SCCmecIII and ST5/SCCmecII [4], [5], and community-associated MRSA (CA-MRSA), which emerged in the community in 1997–1999 with global examples of ST8/SCCmecIV (USA300), ST30/SCCmecIV, ST80/SCCmecIV and ST59/SCCmecV [3], [5], [6], [7]. Hence, globally disseminated ST30/agr3 (but not ST121/agr4) MSSA actually evolved as global CA-MRSA [8].

CA-MRSA possesses distinct virulence factors from those of HA-MRSA, for example, it more strongly expresses cytolytic peptides, such as phenol-soluble modulins (PSMs) [6], [9], [10], and frequently produces Panton–Valentine leucocidin (PVL) [6], [7], [10], [11]. Successful CA-MRSA also has unique virulence factors, as typically shown with USA300 [3], [6], [10].

The elderly are susceptible to S. aureus community-acquired pneumonia (CAP) that requires hospitalization [12], and this may be due to a functionally dysregulated host immune system [13]. Moreover, influenza-infected elderly individuals are at risk of bacterial pneumonia; influenza impairs host immunological mechanisms [14] and promotes co-infections or sequential infections with S. aureus or CA-MRSA [14], [15], [16]. Although PVL is not necessary [16], the pathogenesis of influenza-related CA-MRSA CAP remains unclear.

We herein isolated ST121/agr4 CA-MRSA from influenza-related MRSA CAP for the first time. We then attempted to elucidate the molecular features of CA-MRSA and gain novel insights into the pathogenesis of influenza-related, CA-MRSA-triggered sequential complications.

Case

A 92-year-old man was diagnosed with influenza A using an influenza rapid diagnostic (antigen detection) test on 25 January 2013, and was treated with oseltamivir phosphate (150 mg per day); however, no improvements were noted in his symptoms. He was admitted to a hospital on 27 January 2013 (day 1) with fever and progressive dyspnoea. He had a previous history of cerebral infarction, but did not need regular home visits by healthcare workers and did not have a urinary catheter. He did not have established risk factors for HA-MRSA infections such as a history of hospitalization, surgery, haemodialysis, the presence of a permanent indwelling catheter or percutaneous medical device, or residence in a long-term care facility in the past year [1]. He also had no previous MRSA infections or history of recent antibiotic use. On admission, his white blood cell count and C-reactive protein level were 20,200/μL and 33.3 mg/dL, respectively. Chest radiography revealed bilateral pulmonary infiltrates (Fig. 1): an infiltrative shadow in the right lung (arrow 1) and consolidation with air bronchograms on the lateral side of the left middle lung field (arrow 2). The pattern of the chest radiography findings, a peripherally distributed parenchymal abnormality (arrow 2), is more frequent with MRSA pneumonia than with MSSA pneumonia [17]. Respiratory failure (SpO2, 76%) was noted. Chemotherapy was initiated with a drip infusion of piperacillin (2 g every 12 h). On day 2, sputum was examined, MRSA was detected in cultures (at 107 CFU/mL), and the presence of intracellular MRSA in polymorphs was confirmed microscopically; no other pathogens were detected. On day 5, oral levofloxacin (500 mg every 24 h) was initiated based on the results of drug-susceptibility testing for MRSA (see Supplementary material, Table S1). His respiratory status rapidly improved, and his white blood cell count and C-reactive protein level also improved (4,500/μL and 1.7 mg/dL, respectively) on day 14 (7 February 2013). Hospitalization was continued due to severe anorexia; however, he developed aspiration pneumonia on 11 April and died on 19 April. This case of MRSA, epidemiologically classified as CA-MRSA [1], was named KT1.

Fig. 1

Chest X-ray of the patient on admission day 1. A chest radiograph shows bilateral pulmonary infiltrates. Arrow 1 indicates an infiltrative shadow overlapping the second arch in the middle to lower field of the right lung, while arrow 2 indicates consolidation with air bronchograms on the lateral side of the left middle lung field.

Characterization of microbes

The molecular typing of MRSA, such as sequence type (ST), clonal complex (CC), spa, agr, SCCmec [2], and Coagulase (Coa), was performed as described previously [18]. Forty-nine virulence genes were analysed by PCR [18]: three leucocidin genes (lukSF, lukE-lukD and lukM), five haemolysin genes (hla, hlb, hlg, hlg-v and hld), the peptide cytolysin, PSMα (psmα), 19 staphylococcal superantigen genes, named enterotoxin (SE) or enterotoxin-like (SEl) (tst, sea-e, seg-j, selk-r and selu), staphylococcal exotoxin (set) genes, a staphylococcal superantigen-like gene cluster (ssl), 3 exfoliative toxin (ET) genes (eta/b and etd), the epidermal cell differentiation inhibitor gene (edin), 14 adhesin genes (icaA/D, eno, fib, fnbA/B, ebpS, clfA/B, sdrC-E, cna and bbp), and the arginine catabolic mobile element-arcA gene. MRSA plasmids were analysed as described previously [18]. Bacterial susceptibility testing was performed using the agar dilution method with Mueller–Hinton agar [18]. The mRNA expression level of the PSMα gene (psmα) was examined using an RT-PCR assay [18]. Data were analysed statistically using the Student's t-test. The level of significance was defined as p <0.05.

KT1 exhibited ST121/agr4 and was positive for the enterotoxin gene cluster (egc) with seg, sei, selm, seln, selo and selu [19] (Fig. 2a), similar to global ST121/agr4 MSSA [8]. However, KT1 was negative for PVL, and its spa type (spa1493(t5110)) was divergent from global ST121/agr4 MSSA [8], [20].

Fig. 2

Molecular characteristics (a) and mRNA expression levels of the cytolytic peptide gene (psmα) (b) of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain KT1. In (a), superscript letters indicate the following: athe split hlb gene due to the insertion of phage Sa3; beight adhesin genes are common among S. aureus; cthe 2,473-bp plasmid pWKT1 (GenBank Accession number, LC086373) carried ermC with the leader peptide sequence, responsible for inducible (ind) resistance to clindamycin; pWKT1 was 100% homologous to the pKH19 of MRSA spat1081 (GenBank Accession number, EU350089) and 99% homologous to the pOC160-1 of ST8 CA-MRSA from Russia (GenBank Accession number, AB982226); and daacA-aphD encoded for resistance (r) to gentamicin. (b) Strains KT1 and USA300-0114 are CA-MRSA, and strains N315 and Mu50 are healthcare-associated (HA-) MRSA. The mRNA expression levels of the phenol-soluble modulin α (PSMα) gene (psmα), normalized to 16S rRNA expression levels, were 0.64 ± 0.13, 0.05 ± 0.02, 0.07 ± 0.02 and 0.63 ± 0.13 for strains KT1, N315, Mu50 and USA300-0114, respectively; the psmα expression level of KT1 (no. 1) was similar to that of USA300-0114 (no. 2), and was significantly higher than that of HA-MRSA (p <0.01).

KT1 carried SCCmecV, a characteristic SCCmec of CA-MRSA [2], [3], [5], and oxacillin and imipenem resistance levels were low, which is consistent with CA-MRSA [5], [18]. KT1 carried the characteristic combination of the adhesin genes, cna (for collagen binding) and bbp (for bone sialoprotein binding). Moreover, the PSMα gene expression level of KT1 was significantly higher than that of HA-MRSA (p <0.01), similar to CA-MRSA USA300 (Fig. 2b). Possible ancestral ST121/agr4 MSSA strains also exhibited the same virulence characteristics (see Supplementary material, Fig. S1); those ST121/agr4 MSSA strains (n=14) were PVL+, Coa V, cna+, bbp+, and egc+, and harbored stronger PSMα activities than other MSSA strains [21].

KT1 carried an ermC plasmid (pWKT1), specifying for inducible clindamycin resistance, and the aacA-aphD gene encoding for gentamicin resistance (Fig. 2a; see Supplementary material, Table S1). KT1 was susceptible to generally recommended anti-MRSA agents (see Supplementary material, Table S1).

Discussion

ST121/agr4 MRSA (KT1) met the CDC criteria for CA-MRSA [1]. Moreover, KT1 was bacteriologically consistent with CA-MRSA, based on the SCCmecV carriage [2], [3], [5], SCCmec (mecA) -mediated low level of resistance to oxacillin and imipenem [2], [5], [18], weaker multidrug resistance [5], and strong cytolytic activity [6], [9], [10]. Although elderly MRSA pneumonia in Japan is mostly from ST5/SCCmecII HA-MRSA and, hence, resistant to fluoroquinone [5], MRSA CAP in the present study was a rare fluoroquinone-susceptible case. KT1 exhibited gentamicin and inducible-clindamycin resistance, similar to CA-MRSA in Japan.

We herein described the first case of MRSA CAP from ST121/agr4 CA-MRSA (KT1), originated from PVL+ ST121/agr4 MSSA (including spa type t159), which was globally disseminated in association with skin and soft-tissue infections and necrotizing pneumonia [8], [20]. A unique feature of KT1 over other ST121/agr4 MSSA was PVL–, spat5110 and SCCmecV+.

Influenza impairs host immunological mechanisms [14], promoting the development of CA-MRSA pneumonia [14], [16]. The present case also had influenza-related CA-MRSA CAP, with chest radiography findings showing MRSA pneumonia [17], and was successfully treated with levofloxacin.

We strongly speculate that CA-MRSA associated with CAP harbours unique adhesin and toxic activities that act against the influenza-impaired respiratory tract mucosa. The present case was PVL-independent, which is consistent with previous findings [16]. KT1 (the ST121/agr4 lineage, irrespective of PVL+ or PVL− and MRSA or MSSA) harboured strong adhesin and toxic activities, as characterized by collagen and bone-sialoprotein adhesins, six egc-encoded superantigens, and the strong expression of PSMα. Collagen adhesin is considered to be associated with CAP, possibly through MRSA adherence to damaged tissues, for example, due to viral infection [22]. Globally disseminated ST30/agr3/SCCmecIV CA-MRSA, which has been associated with severe MRSA CAP, also harboured the same adhesin and toxic activities [23], [24].

The present CA-MRSA CAP case, possibly in combination with his previous history of cerebral infarction, developed a decline in the activities of daily living. Pneumonia events have been associated with the loss of physical functioning [25]. In addition, in our patient, CA-MRSA CAP may also have damaged deglutition functions, possibly due to the strong adhesin and cytolytic activities of ST121/agr4 CA-MRSA, resulting in aspiration pneumonia.

ST121 CA-MRSA has been isolated from skin and soft-tissue infections, such as impetigo; however, its genetic characteristics remain unknown [26], [27]; KT1 was negative for impetigo-associated ETs. The ST121/agr4 lineage may be evolving, in two ways, as PVL+, ET+ impetigo-associated CA-MRSA and as PVL– (or PVL+) CAP-associated CA-MRSA.

In conclusion, we isolated ST121/agr4 CA-MRSA (KT1) from influenza-related CA-MRSA CAP for the first time. KT1 originated from globally disseminated PVL+ ST121/agr4 MSSA (with strong adhesin and cytolytic activities), with the unique KT1′ feature of PVL–/spat5110/SCCmecV+. Pneumonia improved with levofloxacin administration, but with the subsequent development of fatal aspiration pneumonia. Hence, professional ST121/agr4 CA-MRSA, with high adhesin and cytolytic activities, triggered influenza-related sequential complications (CAP and aspiration pneumonia).

Conflict of interest

None declared.