Klebsiella pneumoniae (KPN) is a common cause of antimicrobial-resistant opportunistic infections in patients of all ages. KPN could cause a wide range of diseases including pneumonia, urinary tract infections (UTIs), bloodstream infections and sepsis (Effah et al., 2020). These infections are particularly a problem among neonates, elderly and immunocompromised individuals (Bengoechea and Sa Pessoa, 2019). The global emergence and spread of genes of antimicrobial resistance such as ESBL and carbapenemase genes in KPN isolates present a significant danger to public health. High mortality rates, extended hospitalization, coupled with high cost are often associated with infections caused by this organism (Wyres et al., 2020). It was previously thought that KPN generally caused acute infection, but recent studies have found that KPN can exist in the human body for a long time, causing persistent infection or recurrent infection (Zhong et al., 2018; Vachvanichsanong et al., 2020; Collingwood et al., 2023). In this study, KPN was isolated from a child with recurrent pneumonia, and we hypothesized that it was due to the long-term presence of KPN in the child’s respiratory tract leading to recurrent pulmonary infection. However, little research has been done on how KPN persists in the respiratory tract.

Small colony variants (SCVs) are a subgroup of bacteria with special phenotypic and pathogenic characteristics. The most typical feature of SCVs is slow growth, small colony size, and only about 1/10 the size of wild-type strains (Proctor et al., 2006). The first discoverers of SCV were Proctor, who reported the discovery of SCV in Staphylococcus aureus (S. aureus) in 1998 (Proctor et al., 1998). Since then, research on SCVs has been conducted. Further studies have found that many bacteria, such as S. aureus (Von Eiff, 2008), coagulase-negative staphylococci (Fernandez-Rodriguez et al., 2021), Pseudomonas aeruginosa (Anand et al., 2018; Besse et al., 2023), have the ability to form SCVs, and SCVs have been isolated from abscesses, blood, respiratory tract, and soft tissues.

In the process of SCV formation, long-term use of antibiotics and host immune mechanisms play a crucial role. Aminoglycosides, sulfonamides such as compound sulfamethoxazole, quinolones, and other antibiotic can induce the formation of SCVs (Proctor et al., 2006). The intracellular environment of eukaryotic cells also favors the selection and survival of SCVs (Tuchscherr et al., 2011). Increasingly, studies have shown a close association between SCVs and many chronic and recurrent infectious diseases especially SCV of S. aureus (Tuchscherr et al., 2011). Besier et al. showed that the emergence of S. aureus SCVs may be detrimental to the disease process and is related to the deterioration of lung disease in cystic fibrosis patients (Wolter et al., 2013). Compared with the parental strain, in the process of chronic infection, some important exotoxins such as hemolysin and plasma coagulase are significantly down-regulated in S. aureus SCVs, while the synthesis of components related to cell adhesion and biofilm formation is increased (Tuchscherr et al., 2011; Tuchscherr et al., 2015; Guo et al., 2022). Overall, the changes in virulence factor expression in SCVs can affect their ability to cause disease and evade the host immune response. Recent studies have shown that Pseudomonas aeruginosa can also form small colony variants and is closely associated with persistent infections in cystic fibrosis (Besse et al., 2023). Melissa et al. confirmed the causative role of a single ispA mutation in P. aeruginosa SCV emergence and increased aminoglycoside resistance. IspA is involved in the synthesis of ubiquinone, providing a possible link between electron transport and SCV formation in P. aeruginosa (Pitton et al., 2022).

SCV can be classified into two main types: stable and unstable SCVs. Stable SCVs have a stable genetic mutation that results in their small colony size and reduced metabolic activity, while unstable SCVs can revert to the wild-type phenotype under certain conditions. Clinical isolated SCVs often exhibit an unstable phenotype and can easily revert to the parental strain phenotype after passage (Guerillot et al., 2019). In this study, KPN was isolated from a child with recurrent lung infections, and after 48 hours of cultivation, pinpoint-sized colonies were found on blood agar plates. These colonies were identified as KPN by mass spectrometry. In order to maintain the phenotype of clinically isolated SCVs, we added sub-inhibitory concentrations of antibiotics and found that sub-inhibitory concentrations of ciprofloxacin induced the transition of SCV to mucoid-variant of KPN. However, to date, information regarding this phenomenon remains elusive. Better understanding of these data may enable effective treatment of patients at high risk.

The incidence and severity of childhood pneumonia or lower respiratory tract infection (LRTI) have declined substantially in the last decade, but LRTI remains a major cause of mortality in children under five years of age, particularly in low- and middle-income countries (LMICs)(Collaborators, G.B.D.L.R.I, 2018). A retrospective cross-sectional study of 410 South African children hospitalized with KP-bacteremia found that 27% died within 30 days of infection (Buys et al., 2016). Conjugate vaccines (pneumococcal conjugate [PCV], and Haemophilus influenzae type b [Hib] vaccines), have reduced the burden of pneumonia and changed the etiological spectrum (Ning et al., 2017; Pneumonia Etiology Research for Child Health Study, G, 2019; Zar et al., 2022). KPN has been reported to be an important bacterial pathogen that causes neonatal sepsis and mortality, predominantly in southeast Asia (Kurade et al., 2018; Negash et al., 2019; Zar et al., 2022). Even more worrying, recurrent LRTI caused by Klebsiella pneumoniae was also on the rise (Karmaus et al., 2008). Recurrent or persistent infections can expose patients to antibiotics for a long time, this in turn leads to the development of subpopulations of antibiotic persisters (persister cells) that are not susceptible to antibiotics (Kaldalu et al., 2020).

The ability of persisters to survive their interaction with a host is important yet under investigated. Stapels et al. found that Salmonella persisters reprogram macrophages by means of effectors secreted by the Salmonella pathogenicity island 2 type 3 secretion system (Stapels et al., 2018). Another study had reported that Purine metabolism regulates Vibrio splendidus persistence associated with protein aggresome formation and intracellular tetracycline efflux (Li et al., 2023). Ernst et al. demonstrate that the mutants of wzc gene would lead to drug-tolerant K. pneumoniae and could persist to yield potentially untreatable, persistent infection (Ernst et al., 2020). In this study, we isolated SCV from children with recurrent LRTI and speculated that SCV was the reason for the child’s recurring pneumonia.SCV bacteria are commonly found in clinical infections, especially chronic and difficult-to-treat infections such as osteomyelitis, chronic respiratory infections, and cystic fibrosis (Kahl et al., 2016). SCV is a variant form of bacteria that forms smaller colonies on nutrient-rich media. Compared with normal strains, SCV grows slowly and is difficult to detect. Therefore, the epidemiology of SCV bacteria is of great significance. Currently, the epidemiology of SCV bacteria has not been fully studied. It had reported that long-term use of antibiotics may lead to the formation and transmission of SCV bacteria. Antibiotics can kill normal strains, but for SCV bacteria, because of their slow growth, antibiotics may not be able to completely eliminate them, leading to their proliferation in the patient’s body (Proctor et al., 2014; Zelmer et al., 2022). In addition, SCV bacteria may have the ability to adapt to adverse environments, making them easy to transmit in some specific environments(Proctor et al., 2014). Therefore, we speculate that recurrent lower respiratory tract infections in children are associated with the presence of SCV.

At present, the mechanism of SCV formation is still under study. Pitton et al. had confirmed the causative role of a single ispA mutation in SCV emergence and increased aminoglycoside resistance. IspA is involved in the synthesis of ubiquinone, providing a possible link between electron transport and SCV formation in P. aeruginosa (Pitton et al., 2022). Kleinert et al. inserted the IS256 sequence into the wild strain and found a very rapid reversion to the wild type that resembled the fast reversion of clinical SCVs (Kleinert et al., 2017). Islam et al. thought that iron restriction induces the small-colony variant phenotype in Staphylococcus aureus (Islam et al., 2022).

In this experiment, we found that the SCV of KPN had weaker resistance to serum, but was more likely to survive within macrophages. Differential gene analysis showed that the plasmid of the SCV had an additional IcmK family type IV secretion protein compared to the parental strain. The IcmK family type IV secretion proteins are a group of proteins that play important roles both inside and outside of cells. In bacteria, these proteins can be used to inject effector proteins, toxins, and other molecules into host cells, thereby influencing the physiological processes of the host cells(Daveri et al., 2023). Studies have shown that the IcmK family type IV secretion proteins play important roles in the pathogenicity, intracellular survival, and immune evasion of bacteria (Gomez et al., 2013). For example, a study on Pseudomonas aeruginosa found that IcmK family type IV secretion proteins could enhance the survival of this bacteria within host cells, and could also inhibit the apoptotic response of host cells, thereby increasing its virulence (Persat et al., 2015). In addition, Coxiella burnetiid, which causes Q fever, also uses a Dot/Icm-like system to secrete effector proteins via IcmK family type IV secretion proteins. These effectors can modulate host cell processes such as apoptosis, autophagy, and inflammation, allowing the bacterium to establish a replicative niche within the host cell (Segal et al., 2005). Therefore, we speculate that IcmK family type IV secretion proteins may help the SCV of KPN to survive within host cells, thereby enhancing its intracellular survival ability.

In addition, we also found that the SCV of KPN changed to a mucoid colony when exposed to subinhibitory concentrations of ciprofloxacin. A study had reported that Salmonella enterica persister cells form unstable small colony variants after in vitro exposure to ciprofloxacin (Drescher et al., 2019). However, this article does not explain the mechanism. To explain the underlying mechanism of this phenomenon, we conducted whole-genome sequencing analyses of SCV and mucoid colonies.Differential gene analysis revealed that the plasmid of the mucoid-type colony carried DNA polymerase V subunit UmuC gene. UmuC is an important component of the DNA damage-induced mutagenic repair mechanism, and its main function is to participate in the DNA damage repair process (Kuban et al., 2012). When exposed to a DNA damage source, the expression level of UmuC in Klebsiella pneumoniae is upregulated to cope with the DNA damage. As DNA damage repair requires the consumption of cellular energy and resources, and may interfere with normal DNA replication and cell division processes. This may be the reason why the mucoid-type Klebsiella pneumoniae grows slower than the parental strain. In addition, some studies have found that UmuC is quite related to the SOS response, which involved in immune evasion and serum resistance in bacteria (Sutton et al., 2000). Specifically, UmuC may regulate bacterial recognition and evasion of host serum by affecting the surface structure, cell wall composition, and intracellular protein expression of bacteria (Timinskas and Venclovas, 2019; Tanooka et al., 2020). Some studies had demonstrated that umuC gene involved in the formation of tigecycline resistance mechanisms as the potential insertion site of tmexCD1-toprJ1 (Peng et al., 2021; Sun et al., 2022). Although further research is needed to fully understand the specific mechanisms and effects of UmuC on bacterial serum resistance, existing studies suggest that UmuC may be an important regulatory factor in bacterial immune evasion and antibiotic resistance.

Our study showed the SCV of KPN changed to a mucoid colony when exposed to subinhibitory concentrations of ciprofloxacin. Mucoid colonies had a higher resistance of serum, which was possibly related to the UmuC gene. The increased intracellular survival of SCV may be related to the IcmK family type IV secretion proteins.

The data presented in the study are deposited in the NCBI repository, accession number: SAMN36887619, SAMN36887620, SAMN36887621.

The data and samples analyzed in the present study were obtained in accordance with the standards and approved by Chongqing Health Center for Women and Children the Institutional Review Board and Biomedical Ethics Committee. For this study, samples were collected at the microbiology laboratory of our hospital, with no contact with the patient. This study was retrospective and there was no patient identification performed during data collection. Therefore, the ethics committee determined that informed consent was not required.

HZ: Writing – original draft, Writing – review & editing. QL: Methodology, Writing – original draft. JH: Methodology, Writing – original draft. CL: Writing – original draft. YS: Conceptualization, Writing – original draft. LZ: Formal Analysis, Writing – original draft. XZ: Data curation, Writing – original draft.