FormalPara Key Summary Points

Why carry out this study?

 A comprehensive description of M. hominis bloodstream infection (BSI) has rarely been reported. In addition, M. hominis lacks a cell wall that complicates both diagnosis and treatment. As a result, understanding the clinical features, antimicrobial resistance, and pathogenic characteristics of M. hominis BSI is essential for patient care.

What was learned from the study?

 Our results suggest that M. hominis can escalate to sepsis, which has life-threatening consequences. There were no specific clinical symptoms of M. hominis BSI, therefore blood sample culture is critical.

 Antimicrobial-sensitive situations and quinolone-resistant patterns were analyzed, which would guide the empirical selection of antibiotics.

 Nine new alleles and seven new sequence types were described. goeBURST analysis revealed that CC-6 was the main clonal complex of M. hominis sepsis in South China.

Introduction

Mycoplasma hominis is an opportunistic human mycoplasma found in regular bacterial cultures. This pathogen typically colonizes the genitourinary tract, and on rare occasions the respiratory system, in a nonvirulent manner [11]. M. hominis can cause a variety of genital infections, including bacterial vaginosis [26], nongonococcal urethritis, pelvic inflammatory disease, and prostatitis [23]. In recent years, extragenital M. hominis infections have been reported [11, 22, 29]. M. hominis BSI is one of the serious invasive infections caused by this pathogen, and treatment is almost certainly required [18]. However, use antibiotics is severely limited because of the pathogen lacks a cell wall [31]. Fluoroquinolones are commonly utilized as empirical therapy for M. hominis genitourinary infections, and type II topoisomerases, such as DNA gyrase (encoded by the gyrA and gyrB genes) and topoisomerase IV (encoded by the parC and parE genes), interact with fluoroquinolones in M. hominis [4, 8, 38]. However, as shown by irrational drug use, fluoroquinolone resistance in M. hominis has been increasingly problematic.

Many studies continue to focus on M. hominis genitourinary infections; however, the number of reports linked to bloodstream invasiveness is limited. This is, of course, connected to the low frequency or a high prevalence of underdiagnosis of M. hominis from blood. In a Public Health England (PHE) reference laboratory in the UK, only five cases of M. hominis were isolated from blood over a 10-year period [6]. Clinical, drug resistance, and microbiological features of M. hominis BSI are still little known, which might potentially lead to a poor prognosis and even serious clinical crises. A retrospective serial study was conducted, in which the clinical, microbiological, and molecular epidemiological characteristics of eight M. hominis BSI cases from a tertiary teaching hospital in South China were investigated and comprehensively assessed from January 2018 to October 2021. We also examined mutations in quinolone resistance-determining regions (QRDRs). The current study may help us better comprehend M. hominis from blood, as well as provide treatment recommendations.

Methods

Ethical Clearance

Ethical clearance to conduct the study was obtained from Medical Research Ethics Committee at The Second Affiliated Hospital, University of South China (reference number 2021053). The requirement for patient consent to participate in this study was waived because of the retrospective nature of the study. The study followed the latest version of the Helsinki Declaration.

Bacterial Isolates

Between 2018 and 2021, blood samples from all subjects were collected, separated, and cultured in accordance with the Chinese Health Industry Standards. Aerobic and anaerobic bottles (DL Biotech, China) cultures were performed with the use of the BacT/Alert 3D fully automatic blood culture system (BioMerieux, France). The samples from positive blood culture bottles were inoculated onto Columbia blood agar and McConkey plates and incubated at 37 °C in a 5% CO2 atmosphere until visible colonies appeared. According to the routine method of bacterial identification, Gram staining of suspicious colonies was observed.

Clinical strains of M. hominis were screened using the IES kit (Autobio, China) and further identified using real-time fluorescence quantitative PCR (RT-PCR) and 16s rRNA sequencing. For PCR, small-scale preparation of mycoplasmal genomic DNA was performed as previously described [1]. For 16S rRNA sequencing, the following primers were used for PCR amplification: 27f: (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R: (5′-GGTTACCTTGTTACGACTT-3′) [25].

Determination of Antibiotic Resistance

Susceptibility to antibiotics by M. hominis isolates was evaluated using a calibrated 104–105 color-changing units/mL inoculum of each clinical isolate and commercial kits, Mycoplasma IES kit (Autobio, China). Antibiotic susceptibility test results were interpreted according to the manufacturer’s instructions. For the antibiotics in the Mycoplasma kit, which do not include the Clinical and Laboratory Standards Institute (CLSI) breakpoints [33], resistance was determined according to previous studies [13, 19, 23].

Study Subjects

This retrospective study was conducted on patients with M. hominis BSI at The Second Affiliated Hospital, University of South China (Hengyang, China) from June 1, 2018 to October 31, 2021. The diagnosis of M. hominis BSI was based on the Centers for Disease Control and Prevention and National Healthcare Safety Network (CDC/NHSN) definition [12]: (i) the patient has M. hominis cultured from one or more blood cultures and organism cultured from blood is not related to an infection at another site; (ii) the patient has at least one of the following signs or symptoms: fever (> 38 °C), chills, or hypotension. Moreover, signs and symptoms and positive laboratory results were not related to an infection at another site.

Sepsis was defined according to the Sepsis-3.0 criteria [28]: (i) existing evidence of suspected or confirmed infection; (ii) the Sequential Organ Failure Assessment (SOFA) score more than or equal to 2 from baseline or greater than 2 in patients with no baseline score available.

Study Data

Medical records were reviewed to collect the data of patients with M. hominis sepsis during the study period. The study data included the following variables: demographic characteristics (age and sex), underlying or concomitant conditions (diabetes mellitus, kidney disease, hepatic disease, history of intra-abdominal trauma or surgery, malignancy, and hypertension), clinical symptoms, M. hominis treatment time, admission to intensive care unit (ICU), laboratory values (red blood cell [RBC], platelet, white blood cell [WBC] count, percentage of neutrophils, percentage of lymphocytes, percentage of monocytes, C-reactive protein [CRP], procalcitonin [PCT]), urogenital tract operation as cause of hospitalization, and prognosis (cured or died).

Amplification and Sequencing of QRDRs of gyrA, gyrB, parC, and parE Genes

DNA samples were extracted as previously described. Primers for QRDRs of the gyrA, gyrB, parC, and parE genes and the tetracycline resistance gene tetM were designed as previously described [1, 2, 6]. PCR amplification was performed using the following parameters: 30 s at 98 °C, followed by 28 cycles of 20 s at 98 °C, 30 s at 58 °C, and 30 s at 72 °C, and a final extension of 7 min at 72 °C. Positive PCR products were visualized on agarose gel, excised, and sequenced using the Sanger method. Gene mutations and amino acid substitutions of QRDRs were analyzed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the SeqMan software and compared to the reference strain, M. hominis PG21 (ATCC23114 GenBank accession number FP236530.1).

Multiple Sequence Alignment Analysis

BLASTp was performed on the parC and parE protein sequences of M. hominis, and the comparison species were limited to Mycoplasma (taxid: 31969). Nonredundant matched sequences were downloaded, with 1357 sequences (containing gyrA) for parC and 1319 sequences (containing gyrB) for parE. MAFFT software was used to perform a multiple sequence alignment analysis. The amino acid sequences of parC from residues 72 to 151 and of parE from residues 408 to 495 were extracted using M. hominis coordinates to build the sequence logos in WebLogo Version 2.8.2 (http://weblogo.berkeley.edu) [7].

Multilocus Sequence Typing

MLST was performed by sequencing five housekeeping loci (uvrA, GyrB, ftsY, tuf, and gap) [5] according to a previously described method. The M. hominis MLST database (https://pubmlst.org/organisms/mycoplasma-hominis) was used to assign allele numbers and sequence types (STs). The MLST sequence data for the other 59 strains were obtained from Boujemaa et al. [5]. Sequences were aligned, and a phylogenetic tree was constructed using MEGA software version X [16]. The phylogenetic tree was based on concatenated sequences using the neighbor-joining method. Support for internal nodes was estimated using the nonparametric bootstrap method with 1000 replications. Global optimal goeBURST diagrams were drawn up using PHYLOViZ software version 2 [21] to analyze the relatedness between STs.

Results

Clinical Characteristics

The current study examined instances of M. hominis BSI that met the inclusion criteria. Eight strains of M. hominis BSI were collected among the 1148 patients diagnosed with BSI from January 2018 to October 2021 in The Second Affiliated Hospital, University of South China. Table 1 summarizes the clinical features of patients with M. hominis BSI. There were no differences in these instances between men and women, and the median age was 40 (23–81) years. Five patients who suffered severe vehicle accident, cerebral hemorrhage or liver failure developed M. hominis sepsis and were admitted to the ICU, two of whom died. The other three patients, all of whom received urogenital tract surgery, had stable disease, and their body temperature had fallen before M. hominis was identified. Fever was the most frequent clinical symptom (Fig. 1). Before therapy, four patients had WBC levels that were above normal and recovered to normal levels in two patients after treatment. Neutrophils were above the normal range in all patients, whereas lymphocytes and RBCs were below the normal range. The platelets of five patients showed large fluctuations. The mononuclear cells were relatively stable before and after treatment. CPR and PCT in Fig. 1 are the values of the day when a positive blood culture bottle was sent. On the day of diagnosis, all patients had CRP levels above the normal range (13.4–296 mg/L), with only two patients having PCT levels within the normal range. In summary, decreased lymphocyte and RBC counts, increased neutrophil counts, and CRP levels were the most frequently observed.

Table 1 Clinical details of eight patients with M. hominis bloodstream infection
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Fig. 1
figure 1

Body temperature and hematology parameters of patients with M. hominis BSI. a Body temperature; b white blood cells; c neutrophils; d lymphocytes; e monocytes; f red blood cells; g platelets; h C-reactive protein (CRP); i procalcitonin (PCT)

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In terms of clinical care, all patients received empiric antibiotic therapy with β-lactam antibiotics before the infection was identified. However, this effect was not significant. When the pathogen was discovered, tetracyclines or fluoroquinolones were the most frequently used antibiotic regimens. Combination antibiotic therapy was used in two cases (MH-BL01, MH-BL08), with one patient receiving levofloxacin (LEV) combined with doxycycline (DOX) and the other receiving LEV combined with minocycline (MIN). Three patients (MH-BL03, MH-BL04, and MH-BL05) who had not progressed to sepsis showed decreasing trends in temperature and inflammatory parameters before the use of sensitive antibiotics. One patient (MH-BL02) was asked to be transferred to another hospital after being discharged from the ICU, and his follow-up clinical data was not available.

Mutations in Quinolone Resistance-Determining Regions

All isolates were sensitive to DOX, MIN, josamycin (JOS), and clindamycin (CLI). The eight M. hominis strains exhibited resistance to LEV (75.0%), sparfloxacin (SPA, 50.0%), and GAT (37.5%) (Table 2). We performed molecular analysis on seven M. hominis strains, except for one strain that could not be preserved. The seven M. hominis strains, as verified via molecular identification using 16S rRNA sequencing and qPCR (Supplementary Fig. S1), were further amplified for the QRDRs of the gyrA, gyrB, parC, and parE genes. Six types of amino acid substitutions in the QRDRs of the seven strains, based on DNA sequencing, were identified (Table 3): gyrA S153A (one sample) and S153L (five samples), parC S91I (seven samples), K144R (four samples), and parE V417I (seven samples). The double substitution parC S91I/parE V417I was identified in an LEV-sensitive and six LEV-resistant isolates. A mutation in gyrA S153 was detected in six resistant LEV-resistant strains. Four M. hominis strains harbored four substitutions (gyrA S153L or gyrA S153A, parC S91I, parC K144R, and parE V417I), with MICs greater than fourfold increase against SPA and GAT compared with other strains without mutation at parC K144R. Three nucleotide alterations (G1323A, A1347T, and G1428A) (Supplementary Table S2) were identified in the gyrB genes of seven clinical isolates. However, these nucleotide mutations did not cause amino acid changes during translation. Figure 2 provides a graphical representation of amino acid multiple sequence alignment of parC and gyrA and parE and gyrB among Mycoplasma. The overall height of the stack indicates the sequence conservation at this position. The QRDR conservativeness of parC and gyrA was higher than that of parE and gyrB. In the present study, parC and gyrA mutations occurred in a relatively conserved region, which is a hot region of current mutation studies. In comparison, the parC 144 and parE 417 positions were relatively less conserved.

Table 2 Antimicrobial susceptibilities of M. hominis from blood
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Table 3 Antibiotic phenotype and molecular characterization of fluoroquinolone-resistant M. hominis isolates from blood
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Fig. 2
figure 2

Sequence conservation of the quinolone resistance-determining region (QRDR). The logos for parC and gyrA and parE and gyrB were computed for 1357 and 1319 nonredundant sequences, respectively. A logo represents the height of each letter proportional to the observed frequency of the corresponding amino acid. The mutation sites that were based on the M. hominis coordinates are indicated above the symbols

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Multilocus Sequencing Type

MLST revealed seven sequence types among the seven M. hominis strains, including nine new alleles and seven new STs. All STs were first discovered and are shown in Supplementary Table S3. We compared our isolates with clinical M. hominis isolates from other countries by selecting 59 M. hominis isolates from the PubMLST database. Almost all isolates were from the genitourinary tract in Africa (Fig. 3). The 66 human M. hominis isolates were divided into 28 STs. In addition, goeBURST (PHYLOViZ) analysis revealed that one clonal complex (CC) was CC-6 and eight singletons (Fig. 4). CC-6 included 77.3% (51/66) isolates covering 20 STs, and ST6 was defined as the founder of the homologous complex. In this study, five ST types (71.4%, 5/7) belonged to the CC-6 complex group. According to a previous study [5], two major lineages, A and B, were differentiated. The dendrogram generated from the MLST data showed that the majority of STs were grouped into lineage A (18 STs), and it also revealed that sepsis-associated STs were distributed in both lineages.

Fig. 3
figure 3

Neighbor-joining trees of 66 M. hominis isolates based on the concatenated sequences of MLST. The isolates originating from this study are marked in red. aYear of bacteria acquisition

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Fig. 4
figure 4

Phylogenetic analysis. Minimum spanning tree analysis of the 66 M. hominis isolates based on MLST. Isolates are represented by circles, and the size of the circle is proportional to the number of isolates. Branches and numbers represent allelic differences between isolates. Each blue dot represents one ST, and green dots indicate individual founders, while yellow spots denote sub-founders

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Discussion

In previous reports, M. hominis often caused maternal postpartum or postabortal fever and often settles without treatment [32], and immune functions play an essential role in regulating M. hominis sepsis [9]. In the immunocompetent host, M. hominis can be efficiently cleared from the circulation by phagocytes and macrophages in the blood and the reticuloendothelial system [9]. In our study, those patients who had a clear urogenital source presented with a transient BSI and both of them required irrelevant antibiotics and survived, whereas patients admitted with major trauma to ICU progressed to sepsis and needed effective therapy for a longer time but nevertheless in some cases died. Although most M. hominis BSI have been transitory in prior research [34], the possibility of progression should not be discounted. This could be related to the patient’s immunological function, although it was not investigated in this study.

Several elements of the clinical presentation of M. hominis sepsis were validated via our investigation (Fig. 1). The most common clinical features were fever. Additional findings included lymphopenia, increased neutrophil count and CPR, and only two patients having PCT levels within the normal range. Furthermore, although all our patients had erythropenia, there is no evidence to support a link between this clinical presentation and M. hominis BSI. When compared with patients who had BSI caused by other bacteria of the same period, no significant differences in temperature, CRP, PCT, WBC, neutrophil count, platelet and RBC were observed (P > 0.05) (Supplementary Table S1), which suggested that there were no characteristic changes in inflammatory indicators of M. hominis BSI. Therefore, it is extremely important to analyze blood cultures from patients with severe infections, and those who have failed to respond to β-lactams should be warned about the risk of M. hominis BSI. (The inclusion criteria and results of statistical analysis for the control group are described in the Supplementary Material.)

M. hominis is inherently resistant to all β-lactams because of the lack of a cell wall. To further limit treatment choices, two antibiotic families, fluoroquinolones and tetracyclines, showed strong action against these bacteria [3]. In antibiotic susceptibility studies of M. hominis in the urinary tract, a German study [15] showed that ciprofloxacin (CIP) may be used in empirical treatment; CLI and JOS could be used as alternatives. Italian [17] and Hungary studies [24] also showed low resistance to fluoroquinolones. A lower sensitivity to JOS (79.2%) for M. hominis in Athens has been reported [14]. In a study in Beijing, the resistance rates of M. hominis to CIP, LEV, and SPA were 82.2%, 62.2%, and 80.0%, respectively, and few strains showed resistance to MIN, DOX, and JOS [35]. These results were similar to those of some other studies in China [37,38,39] and current research. As a result, the antibiotic sensitivity profile of M. hominis isolated in various regions of the genitourinary tract can be utilized as a reference for the treatment of M. hominis BSI. In China, MIN, DOX, or JOS could be used as empirical therapy for M. hominis BSI.

The predominance of reduced susceptibility to fluoroquinolones among M. hominis isolates is an alarming sign. Sequence analysis of QRDRs in LEV-resistant M. hominis strains indicated that parC S91 and gyrA S153 were mutation hotspots (Table 3). Previous research has found that these two mutations are the most common genetic changes in fluoroquinolone-resistant M. hominis [8, 20, 38]. Furthermore, these two positions exhibited considerably higher conservation (Fig. 2). parC S91 (corresponding to mutations in Ureaplasma spp. parC S83) substitution was the most frequent mutation in Ureaplasma spp. [27, 30]. However, a mutation in parC S91I was observed in one of the bacterial strains susceptible to LEV. This might be because mutations in gyrA S153 constitute the major mechanism of LEV resistance in clinical isolates of M. hominis, whereas changes in parC S91 allow resistance to increase to a greater degree. Similar findings have been previously published in the literature. According to Zhang et al. [38], a single parC S91I mutation increased the MIC of LEV from 0.5 to 4–8 g/mL. The MIC of LEV was enhanced to 128 g/mL when ParC S91I was coupled with gyrA S153L. According to Yang et al. [36], strains carrying the two mutations had a two- to fourfold higher MIC for LEV than those carrying parC S91 alone. Structural analysis of the wild-type topoisomerase IV (ParC–ParE) complex coupled to LEV in Ureaplasma spp. revealed that parC Ser83 or its analogous mutations in M. hominis (gyrA S153A, gyrA S153L, and parC S91I) precluded interactions with LEV. Controversially, some studies have indicated that the ParC K144R mutation does not contribute to fluoroquinolone resistance [8, 36, 38], while Meng et al. [19] indicated that this mutation point substitution in parC of M. hominis might be related to its resistance to ofloxacin and LEV. This mutation site was also discovered in our clinical isolates, but it appeared to increase the MIC values of SPA and GAT. Because a single mutation was found in sensitive strains in prior research, it is possible that K144R with parC S91 and/or gyrA S153 might promote resistance only when present concurrently, although the biological relevance of these modifications remains unknown. We discovered that all isolates had identical ParE aberrations that resulted in an amino acid exchange (Val 417 Ile) compared to the type strain PG21. This might be related to genetic variation. Several nucleotide changes occurred in the gyrB gene, consistent with previous results [10, 20] and our findings; however, these were nonsense mutations with no amino acid change.

MLST analysis revealed the molecular epidemiological characteristics of M. hominis. We also highlighted the higher genetic diversity of sepsis-associated M. hominis in South China. We identified a total of seven STs, including seven new STs, which means all of the STs were the new types. Because the data we jointly evaluated were only from genitourinary tract isolates from one hospital in North Africa, it is unknown if this genetic diversity is related to the area or the causal location, and it is worth additional investigation when the database is richer. CC-6 was not only the main CC of M. hominis isolated from the genitourinary tract in North Africa but was also isolated from the blood. CC-6 may be more closely associated with BSI. In addition, the dendrogram generated from the MLST data in this study showed that the majority of STs were grouped into lineage A (57.1%, 4/7), and our strains were not concentrated in a particular lineage or formed a new lineage. However, none of the MLST-derived data could be correlated with the year and source of isolation of the clinical strains.

Our study had a few limitations. First, the extremely limited instances and strains are a potential drawback of our investigation. At the same time, however, few similar cases of this kind have been reported, so we hope that our report will provide some help in clinical management. Second, because this study was retrospective, the inconsistency of the clinical data prevented further utilization and analysis of more significant indicators.

Conclusions

A serial study of M. hominis bloodstream infection was conducted. Substantial levels of fluoroquinolone resistance in M. hominis were found in South China. It is critical to promptly identify the features of M. hominis sepsis, followed by effective therapeutic strategies. Furthermore, more research is necessary to confirm the connections between the genotypes, resistant spectrum, and clones of M. hominis isolates from blood. Further studies are required to improve the awareness of M. hominis and to develop effective therapies for patients with M. hominis sepsis.