*Article* **Genomic Analysis of Multidrug-Resistant Hypervirulent (Hypermucoviscous)** *Klebsiella pneumoniae* **Strain Lacking the Hypermucoviscous Regulators (***rmpA***/***rmpA2***)**

**Hisham N. Altayb 1,2,\* , Hana S. Elbadawi <sup>3</sup> , Othman Baothman <sup>1</sup> , Imran Kazmi <sup>1</sup> , Faisal A. Alzahrani 1,2,4 , Muhammad Shahid Nadeem <sup>1</sup> , Salman Hosawi 1,2 and Kamel Chaieb 1,5**


**Abstract:** Hypervirulent *K. pneumoniae* (hvKP) strains possess distinct characteristics such as hypermucoviscosity, unique serotypes, and virulence factors associated with high pathogenicity. To better understand the genomic characteristics and virulence profile of the isolated hvKP strain, genomic data were compared to the genomes of the hypervirulent and typical *K. pneumoniae* strains. The *K. pneumoniae* strain was isolated from a patient with a recurrent urinary tract infection, and then the string test was used for the detection of the hypermucoviscosity phenotype. Whole-genome sequencing was conducted using Illumina, and bioinformatics analysis was performed for the prediction of the isolate resistome, virulome, and phylogenetic analysis. The isolate was identified as hypermucoviscous, type 2 (K2) capsular polysaccharide, ST14, and multidrug-resistant (MDR), showing resistance to ciprofloxacin, ceftazidime, cefotaxime, trimethoprim-sulfamethoxazole, cephalexin, and nitrofurantoin. The isolate possessed four antimicrobial resistance plasmids (*p*KPN3-307\_type B, *p*ECW602, *p*MDR, and *p*3K157) that carried antimicrobial resistance genes (ARGs) (*bla*OXA-1, *bla*CTX-M-15, *sul2*, *APH(3*00*)-Ib*, *APH(6)-Id*, and *AAC(6*0 *)-Ib-cr6*). Moreover, two chromosomally mediated ARGs (*fosA6* and *SHV-28)* were identified. Virulome prediction revealed the presence of 19 fimbrial proteins, one aerobactin (*iutA*) and two salmochelin (*iroE* and *iroN*). Four secretion systems (T6SS-I (13), T6SS-II (9), T6SS-III (12), and Sci-I T6SS (1)) were identified. Interestingly, the isolate lacked the known hypermucoviscous regulators (*rmpA/rmpA2*) but showed the presence of other *RcsAB* capsule regulators (*rcsA* and *rcsB*). This study documented the presence of a rare MDR hvKP with hypermucoviscous regulators and lacking the common capsule regulators, which needs more focus to highlight their epidemiological role.

**Keywords:** antimicrobial resistance; hvKP; K2 capsule; ST14; fimbrial proteins; aerobactin

#### **1. Introduction**

*Klebsiella pneumoniae* is a Gram-negative bacterium associated with invasive hospitalacquired infections [1]. Hypervirulent *K. pneumoniae* (hvKP) overproduces a polysaccharide capsule and is an important clinical pathogen responsible for several infections in healthy and immunosuppressed patients [2,3]. The presence of capsular polysaccharides (CPS) and lipopolysaccharides (LPS) are associated with organism dissemination and virulence [4].

**Citation:** Altayb, H.N.; Elbadawi, H.S.; Baothman, O.; Kazmi, I.; Alzahrani, F.A.; Nadeem, M.S.; Hosawi, S.; Chaieb, K. Genomic Analysis of Multidrug-Resistant Hypervirulent (Hypermucoviscous) *Klebsiella pneumoniae* Strain Lacking the Hypermucoviscous Regulators (*rmpA*/*rmpA2*). *Antibiotics* **2022**, *11*, 596. https://doi.org/10.3390/ antibiotics11050596

Academic Editor: Teresa V. Nogueira

Received: 21 March 2022 Accepted: 26 April 2022 Published: 28 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

This pathotype with hypermucoviscosity has acquired antimicrobial resistance capable of causing serious invasive disease, unlike the old drug-susceptible strains [3]. The presence of hvKP has been linked to endophthalmitis, pneumonia, liver abscesses, and meningitis [5]. The hvKP phenotype, which contributes to the hypermucoviscous phenotype, is related to the presence of a virulence plasmid containing two capsular polysaccharide regulator genes (*rmpA* and *rmpA2*) as well as multiple siderophore gene clusters and capsular K antigens (*K1*, *K2*, *K5*, *K20*, *K54*, and *K57*) [6,7]. Most of the hvKPs belong to a small collection of clonal groups; the more dominant groups are CG23 and include ST23, 26, 57, and 1633 [8].

Capsules, siderophores, lipopolysaccharides (LPS), fimbriae, outer membrane proteins, and type 6 secretion systems (T6SS) are among the virulence components that contribute to hvKP strains [9]. Most of the hypermucoviscous and hypervirulent strains of *K. pneumoniae* are characterized by the presence of the *rmpA* and *rmpA2* (transcriptional activators, which regulate the mucoid phenotype) regulatory genes [10], but in a few cases, these strains could lack the *rmpA* and *rmpA2* regulators [8,11].

Aerobactin is considered one of the most critical virulence factors in hvKP and is used for the definition of hypermucoviscous strains such as hvKP [6]. Aerobactin-producing isolates are more likely to cause a severe immune response in the host and more invasive infections [6]. In Taiwan, hypermucoviscosity was seen in 88.8% of *K. pneumoniae* isolates from individuals with pyogenic liver abscesses [12]. A purulent liver abscess caused by a very invasive community-acquired *K. pneumoniae* has recently been reported [3]. Furthermore, an outbreak of ST11-type carbapenem-resistant hvKP was reported in a Chinese hospital in 2016 [13].

Most of the hvKPs have remained susceptible to a variety of routinely used antimicrobial agents with the exception of ampicillin, but recently MDR isolates have been increasingly reported worldwide [14–16]. Carbapenem-resistant *K. pneumoniae* strains from the clonal group (CG) 258 are the most prevalent, with ST258 and ST11 being the most common multilocus sequence types globally [17]. The acquisition of virulence plasmids by *K. pneumoniae* harboring the insertion of the drug resistance genes *bla*KPC-2 and *catA1* has been reported [18,19]. According to Hao et al. [3] the rates of the virulence-associated genes *rmp*A, *iro*B, *fib*, and *hib* were considerably greater in hvKP than in non-hvKP. Furthermore, plasmids carrying two replicons (IncHI1B–IncFIB and IncFIIK–IncFIBK) coding for drugresistant and virulence genes were discovered [20,21]. The presence of a wide range of β-lactamases, aminoglycoside, and carbapenem-resistant genes could result in the increasing difficulty of treatment and long hospital stays [16,22]. More recently, hvKP belonging to ST147 in COVID-19 patients has been reported in Italy with three plasmid replicons of the IncFIB (Mar), IncR, and IncHI1B types as well as different resistance genes [23]. Additionally, fourteen colistin-resistant *K. pneumoniae* (CoRKp) strains were screened retrospectively in China between 2017 and 2018 [24]. Among them, six CoRKp strains belonging to ST11 were MDR [24].

Khartoum is one of the most crowded cities in Africa [25,26] which facilitates the horizontal transfer of antimicrobial-resistant bacteria. Additionally, Sudan suffers from the inappropriate use of antibiotics; most of the antibiotics are frequently sold over the counter and even without a medical prescription [27,28]. In a recent study conducted in Khartoum state, strains positive for β-lactamase and carbapenemase genes have been reported in hvKP isolates [29]. To better understand the genomic characteristics and virulence profile of the newly isolated hvKP strain (named 9KP), this comparative genomic study was conducted.

#### **2. Results**

#### *2.1. Patient Details and Phenotypic Characterization of the Isolate*

The isolate was obtained from a patient with CKD in Soba University Hospital in Sudan, and it was identified with a hypermucoviscous phenotype using the string test, in which mucus is measured more than 9 cm by lop (Supplementary file 1, Figure S1). The isolate was classified according to CLSI breakpoints as MDR when showing resistance to ciprofloxacin, ceftazidime, cefotaxime, trimethoprim-sulfamethoxazole, cephalexin, nitrofurantoin, amoxicillin-clavulanic acid, and ampicillin, while it was susceptible to meropenem, imipenem, amikacin, and gentamicin. A high resistance level was observed for cephalosporins and penicillin, in which a no inhibition zone (0 mm) was observed for amoxicillin-clavulanic acid and ampicillin. Additionally, for the first-generation and third-generation cephalosporins, a small zone of inhibition (10 mm) was observed. Among non-β-lactams, a high resistance level was observed for trimethoprim-sulfamethoxazole (0 mm) and a small zone of inhibition (10 mm) was observed with ciprofloxacin (Table 1).


**Table 1.** Antimicrobial susceptibility testing of selected antimicrobial agents used against 9KP strain.

Abbreviation: R = Resistant, S = Sensitive, - = Not tested, mm = millimeter; <sup>a</sup> Antimicrobial susceptibility testing determined according to CLSI guidelines [30].

For the determination of the minimum inhibitory concentrations (MIC) of the antibiotics, we used the microtitre broth dilution method, which revealed that the isolate possessed a high resistance level against ampicillin (MIC = 1024 µg/mL), tetracycline (MIC = 256 µg/mL), cefotaxime (MIC = 128 µg/mL), and ciprofloxacin (MIC = 128 µg/mL), while two antimicrobial (gentamicin and chloramphenicol) scored a very low MIC (4 µg/mL), falling within the susceptibility range according to CLSI guidelines [30] (Table 1).

#### *2.2. Genome Characteristics and Typing*

The total genome was assembled into 5364730 bp, with 83 contigs and an average contig length of 64635, while N50 was 220979, L50 7, the average coverage was 100X, and the GC content was 57.3%. The total number of predicted genes was 5248, 76 tRNA, and 202 genes associated with stress response, defense, and virulence (Supplementary file 1, Figure S2). The isolate was identified as *K. pneumoniae* with sequence type (ST) 14 by the Institut Pasteur MLST and MLST 2.0 databases. The global platform for genomic surveillance, Pathogenwatch, was used for the prediction of the capsule (K) and O serotypes; the isolate was identified with the K2 (wzi2 genotype) capsule and O1 serotype. The 9KP strain harbored ten antimicrobial resistance genes including β-lactam resistance genes (*bla*OXA-1, *bla*CTX-M-15, and *bla*SHV-28), sulfonamide resistance (*sul2*), fosfomycin resistance (*fosA6*), aminoglycoside resistance (*APH(3*00*)-Ib*, *APH(6)-Id*, and *AAC(6*0 *)-Ib-cr6*), and the gene causing resistance to tetracycline (*tet(A)*). The chloramphenicol O-acetyltransferase (*CatB3*) gene was detected in the 9KP strain with 70% coverage and 100% identity (Supplementary file 1, Table S1). Additionally, three efflux pumps were identified, including *K. pneumoniae KpnF, LptD,* and *oqxA*. Two chromosomal mutations conferring resistance to fosfomycin (E350Q) and

elfamycin EF-Tu (R234F) were also identified. The PlasmidFinder tool revealed the presence of four plasmid replicons (Col440II, IncFII, IncFIB(K), and IncFII(K)) in the 9KP strain with 100% identity and coverage. Additionally, the use of a BLASTn search against the PLSDB database revealed the presence of four plasmids in the 9KP strain, carrying different ARGs, *p*KPN3-307\_type B, *p*ECW602, *p*MDR, and *p*3K157, which showed a matching of 99.56%, 99.75%, 100%, and 100%, respectively. The *p*KPN3-307\_type B plasmid of the *K. pneumoniae* strain H151440672 was identified in our strain as carrying genes corresponding to *bla*CTX-M-15, RND efflux, and IS1 sequences (Supplementary file 1, Figures S3 and S4). The *Escherichia coli* plasmid *p*ECW602 was detected in the 9KP strain carrying different mobile elements and ARGs-encoding genes, which included sulfonamide (*sul2*) and aminoglycoside resistance genes (*APH(3*00*)-Ib* and *APH(6)-Id*) (Figure 1). *K. pneumoniae p*MDR was identified with two transposases capturing *tet(A)* MFS-family efflux-pump-encoding genes (Supplementary file 1, Figure S5). Moreover, we detected the chloramphenicol Oacetyltransferase (*CatB3*) gene, class D beta-lactamase (*bla*OXA-1), and aminoglycoside N(60 ) acetyltransferase (*aac(6*0 *)-Ib-cr*) genes in the 9KP plasmid (*p*3K157) (Supplementary file 1, Figure S6) while *SHV-28* and fosfomycin resistance (*fosA6*) genes were detected only in chromosomal sequences and were absent in the assembled plasmid, indicating their possible chromosomal association.

One plasmid belonging to the IncFIB(K) type was identified by a BLASTn search against PLSDB and showed 99.7% identity to the *K. pneumoniae* strain SCKP020143 plasmid *p*1\_020143, and it was negative for ARGs (Supplementary file 1, Figure S7).

The virulence factor database was used for the prediction and comparison of the virulence genes of the 9KP strain with others. Different types of fimbrial proteins were discovered including type I (10), type 3 (8), and type IV pili (*pilW*) (Table 2) (Supplementary file 1, Table S2). A total of 15 iron uptake proteins were identified, including 1 aerobactin (*iutA*), 12 Ent siderophores, and 2 salmochelin, while it lacked the other aerobactin (*iucA*, *iucB*, *iucC*, and *iucD*) reported in the hvKP strains (NTUH-K2044 and KCTC 2242). The most closely related strains (kkp066 and kkp0e7) were positive for the hvKP marker, the *RmpA* gene, and lacked aerobactin (*iucA*, *iucB*, *iucC*, and *iucD*), similar to our strain. High similarity in the iron uptake system of 9KP and the other Sudanese strain (23KE) was observed, including the complete absence of genes related to yersiniabactin and the presence of two salmochelin and one aerobactin. Four secretion systems that are crucial virulence factors of pathogenic bacteria were identified in the 9KP strain, including T6SS-I (13), T6SS-II (9), T6SS-III (12), and one Sci-I T6SS exclusively detected in our strain. The isolate was positive for two *RcsAB* (*rcsA* and *rcsB*) regulatory proteins and one serum resistance LPS protein. The mediator of the hyper adherence *YidE* in enterobacteria and its conserved region were predicted in the isolate.

*Antibiotics* **2022**, *11*, 596


Key: + means the presence of the same number of genes, - means gene absent, numbers in tables indicate numbers of virulence-factors-related genes.

**Figure 1.** Linear map of *E. coli* plasmid *p*ECW602 which was detected in 9KP strain; the horizontal black lines indicate the length of the plasmid, the middle gray line contains information about plasmid length and coverage. In addition, the purple arrows indicate mobile elements and hypothetical proteins. The green arrows indicate ARGs. **Figure 1.** Linear map of *E. coli* plasmid *p*ECW602 which was detected in 9KP strain; the horizontal black lines indicate the length of the plasmid, the middle gray line contains information about plasmid length and coverage. In addition, the purple arrows indicate mobile elements and hypothetical proteins. The green arrows indicate ARGs.

**Table 2.** Comparison of virulence factors of *K. pneumoniae* 9KP with other control strains (*K. pneumoniae* 342, MGH 78578, NTUH-K2044, 1084,

HS11286, JM45, KCTC 2242, SB3432) and the most related strains (kkp066, kkp0e6, and 23KE).

**Virulence Factor Related Genes 9KP 342 MGH78578 NTUH-K2044 1084 HS11286 JM45 KCTC 2242 SB3432 kkp066 kkp0e6 kkp0e7 23KE**

Adherence

Antiphagocytosis

Efflux pump

Iron uptake

Nutritional factor

Capsule 1 + + + + + + + + + + + + +

AcrAB 2 + + + + + + + + + 1 + + +

Aerobactin 5 1 1 1 + 1 1 1 + + 1 1 1 1 Ent siderophore 13 12 + + + + + 12 + - 12 10 11 + Salmochelin 5 2 2 2 + 4 2 2 2 4 2 2 2 2 Yersiniabactin 11 - - - + + + - - - - + + -

Type 3 fimbriae 8 + + 7 + + + + + + + + 7 + Type I fimbriae 10 + + + + + + + + + 9 9 8 + Type IV pili 12 1 - - - - - - - - - - - -

#### *2.3. Comparative Genomics and Phylogenomics Analysis*

After the genome comparison, the species formed 6142 protein clusters, 3185 orthologous, and 2957 single-copy gene clusters. 9KP showed 192 single-copy genes and 4843 proteins clustered with others (Supplementary file 1, Table S3). A high degree of variability was observed at different chromosomal regions of 9KP, which contains ARGs, incF plasmid proteins, IS, and other mobile elements.

Comparative genomics revealed that the strains TCC BAA-2146, 23KE, kkp066, kkp0e6, and NTUH-K2044 exhibited a high similarity to 9KP, in which different virulent regions were similar, such as the outer membrane protein OmpN, LysR-*type* transcriptional regulators, kinase, and fimbrial proteins (Figure 2) detected at a region located between the chromosomal range 1.5–1.6 Mb. Ferric enterobactin-related proteins and phage-related proteins were clustered in *K. pneumoniae* 9KP similarly to the strains ATCC BAA-2146 and NTUH-K2044 (Figure 3), while the secretion systems T6SS were located in a region adjacent to the VgrG protein, transposases, putative kinase, mobile elements, transcriptional regulator, LysR family, and phage proteins. The PTS system in the 9KP strain was most similar to the PTS system of the 23KE strain from Sudan others (Supplementary file 1, Figure S3, and Supplementary file 2).

A phylogenetic tree was generated among the African strains by the iTOL—Interactive Tree of Life—*Klebsiella* Pasteur MLST database. The 9KP strain was clustered in a clade containing three strains from Kenya, one was isolated from a patient with a soft tissue infection (kkp066) and the others (kkp0e6 and kkp0e7) were isolated from hospital environment. And it was also clustered to one MDR Sudanese strain (K23) isolated from drinking water in Khartoum state (Figure 4).

**Figure 2.** Clustering of fimbrial proteins in contig 7 of *K. pneumoniae* 9KP; the horizontal black lines indicate the contig length, the green arrows indicate genes **Figure 2.** encoding fimbrial proteins, and the purple arrows indicate other genes located at the same contig. Clustering of fimbrial proteins in contig 7 of *K. pneumoniae* 9KP; the horizontal black lines indicate the contig length, the green arrows indicate genes encoding fimbrial proteins, and the purple arrows indicate other genes located at the same contig.

**Figure 3.** Clustering enterobactin proteins in contig 32 of *K. pneumoniae* 9KP; the horizontal black lines indicate the contig length, the green arrows indicate genes **Figure 3.** Clustering enterobactin proteins in contig 32 of *K. pneumoniae* 9KP; the horizontal black lines indicate the contig length, the green arrows indicate genes encoding enterobactin proteins, and the purple arrows indicate other genes located at the same contig.

encoding enterobactin proteins, and the purple arrows indicate other genes located at the same contig.

**Figure 4.** Phylogenomics analysis of *K. pneumoniae* 9KP (shown in red highlight) compared to African *K. pneumoniae* strains. Strain 3KE is *K. quasipneumoniae* used as an outgroup. Numbers in nodes indicate Pasteur MLST isolate IDs. **Figure 4.** Phylogenomics analysis of *K. pneumoniae* 9KP (shown in red highlight) compared to African *K. pneumoniae* strains. Strain 3KE is *K. quasipneumoniae* used as an outgroup. Numbers in nodes indicate Pasteur MLST isolate IDs.

#### **3. Discussion**

Hypervirulent *K. pneumoniae* strains possess distinct morphological and genotypic characteristics when compared to other classical strains, which include the production of colonies with hypermucoviscosity, unique serotypes, and virulence factors associated with high pathogenicity [31]. Except for ampicillin, most of the hvKPs have remained susceptible to a variety of routinely used antimicrobial drugs, but recently MDR isolates have been increasingly reported worldwide [14–16]. The present study reported MDR hvKP in a patient with a recurrent UTI, and it harbored genes conferring resistance to β-lactam (*bla*OXA-1, *bla*CTX-M-15, and *bla*SHV-28), sulfonamide (*sul2*), fosfomycin (*fosA6*), and aminoglycoside (*APH(3*00*)-Ib, APH(6)-Id*, and *AAC(6*0 *)-Ib-cr6*). The presence of a wide range of β-lactamases and aminoglycoside-resistant genes could result in the increased difficulty of treatment and long hospital stays [16,22]. *Klebsiella* species are known to have intrinsic resistance to ampicillin [32], and here we reported a very high resistance level to ampicillin (MIC ≥ 1024 µg/mL). This could be a result of the presence of additional beta-lactamases (*bla*CTX-M-15, *bla*OXA-1, and *bla*SHV-28). A high resistance level was also observed against cefotaxime (MIC ≥ 128 µg/mL), which could be attributed to the presence of *bla*CTX-M-15 which possesses a high hydrolytic activity against cefotaxime [33]. Although the isolate harbored chloramphenicol O-acetyltransferase (*CatB3*), the isolate was highly susceptible to chloramphenicol. This could be due to the truncation of the gene, which only showed 70% coverage to the references.

Our isolate harbored an IncF plasmid, insertion sequences, and phage-associated proteins at regions containing ARGs and virulence genes, which reflect their possible role in the horizontal gene transfer and dissemination of such strains [16]. The IncF plasmids are thought to play a significant role in the acquisition of MDR genes [34,35], which could increase the chance for the acquisition of genes such as the *bla*KPC carbapenem resistance gene.

We identified four plasmids that carried different ARGs and transposases. The presence of the ARGs plasmids in the hvKP strain, which is known to be a more drugsusceptible strain [36], could be a reason for the presence of the MDR phenomenon in our isolate. Additionally, these plasmids may result in the mobility of these ARGs to drug-susceptible isolates.

Our isolate harbored a *p*KPN3-307\_type B plasmid that carried genes corresponding to *bla*CTX-M-15, RND efflux, and IS1 sequences; similar plasmids carrying *bla*CTX-M-15 with transposases have been reported in the KPC-producing *K. pneumoniae* ST307 strain in the UK [37]. The presence of the *CTX-M* gene in the mobile elements could be the reason for the current dissemination of the *CTX-M*-positive isolates in our region [38,39]. Moreover, the isolate possessed the heavy metal (copper(I)/silver(I)) efflux pump (RND efflux); isolates resistant to silver have more affinity to establishing hospital and environmental outbreaks [40]. Interestingly, the 9KP strain harbored the plasmid *p*ECW602, which is a novel plasmid reported recently in an extensively drug-resistant (XDR) *E. coli* isolate in China [41]; here we reported it for the first time in a *K. pneumoniae* (9KP) isolate with high identity (99.75%) and high coverage (744). The 9KP plasmid (*p*ECW602) and *E. coli p*ECW602 plasmid carried a similar pattern regarding the presence of sulfonamide (*sul2*) and aminoglycoside resistance genes (*APH(3*00*)-Ib* and *APH(6)-Id*). The gene responsible for the resistance to tetracycline (*tetA*) associated with the MFS family efflux pump was identified in the *K. pneumoniae* 9KP strain *p*MDR plasmid; the gene expression of the MFS-type *tetA* has been documented in different Gram-negative isolates [42,43]. tet(A) bearing K. pneumoniae was reported with a high tetracycline and tigecycline resistance level [42]. Adding to that, another tetracycline resistance efflux (*oqxA*) was discovered in our isolate [44]. In addition to plasmid-mediated ARGs, two genes (*fosA6* and *SHV-28)* were not detected among the assembled plasmids of the 9KP strain but they were present in the chromosomes; the fosfomycin resistance gene (*fosA6)* and the broad spectrum B-lactamase *SHV-28* gene are commonly reported in *K. pneumoniae* chromosomes [45–48].

The isolate lacked the common regulators of the hypermucoviscous phenotype *(rmpA*/ *rmpA2*) [49] and yersiniabactin system but showed the presence of aerobactin-(*iutA*) and salmochelin-(*iroE* and *iroN*) encoding genes, which are clear markers for hvKP identification [50]. Additionally, the strain was predicted with the K2 capsule type and hypermucoviscosity, which are common virulence factors in hvKP [51]. Similarly, strains belonging to hvKP and lacking the *rmpA* and *rmpA2* genes were previously reported without knowledge of the mechanisms of capsule overexpression [52,53]. One possible explanation of the mucoviscosity in *K. pneumoniae* 9KP is the presence of the *RcsA* and *RcsB* genes; the *RcsA* gene binds with *RcsB* to activate the genes responsible for capsular polysaccharide production in *E. coli* [54]. Another explanation for the presence of the siderophore receptors without biosynthetic genes in hvKP is that these strains can acquire the siderophores from other bacteria found in the same environment [8]. Similar to our finding, a highly virulent and invasive *K. pneumoniae* strain possessing genes such as aerobactin (*iutA*), hypermucoviscosity, salmochelin, and lacking *rmpA*/*rmpA2* was reported in a patient suffering from necrotizing soft tissue infection at Northwestern Memorial Hospital, USA [51].

In this study, four T6SS systems were detected. The type VI secretion system (T6SS) is usually located at the chromosomes or pathogenicity islands of virulent bacteria, and they have a role in host infection and colonization [55]. Additionally, eight type 3 fimbrial proteins were reported. Usually, isolates that express type 3 fimbriae are more biofilmproducing compared to other strains [56]. Biofilm-producing isolates can cause community or hospital infections and are associated with 65% of microbial infections and 80% of chronic infections globally [57]. Furthermore, the genomic analysis of the *K. pneumoniae* 9KP strain demonstrated a large abundance of LysR-family transcriptional regulators in the genomic regions containing a cluster of virulence and antimicrobial resistance genes. *LysR* is found in different bacterial species and has a role in the regulation of virulence factors in pathogenic bacteria [58]. A novel type of the LysR family has been demonstrated to have a pleiotropic role in mediating the resistance and increasing the virulence of the hvKP NTUH-K2044 strain [59].

The phylogenetic analysis showed that the 9KP strain is more related to strains from Kenya and Sudan. This could be due to the fact that Kenya is a neighboring country to Sudan, and the Sudanese clustered isolate was from the same location (Khartoum) of the sample collection in our study. Two of the Kenyan strains (kkp066 and kkp0e7) were hvKPs possessing the *RmpA* gene and lacked aerobactins (*iucA*, *iucB*, *iucC* and *iucD*), similar to our strain. Additionally, the 9KP strain showed a high similarity in the PTS system to the 23KE strain from Sudan. This could be one of the reasons behind their high similarity to our strain.

MDR and hvKP strains previously developed in distinct clonal groups [60] but the recent emergence of hvKP isolates carrying MDR genes needs more attention. Such a strain has the potential to produce fatal hospital outbreaks, so more focus is needed to highlight its epidemiological role.

#### **4. Methods**

#### *4.1. Bacterial Isolation, Identification, Susceptibility Testing, and DNA Extraction*

*Klebsiella* spp. was isolated from the urine sample of a 40-year-old male patient with a history of recurrent UTI, hypertension, and chronic kidney disease (CKD) admitted for hemodialysis in Soba Hospital, Khartoum in July 2021. The patient was visiting the dialysis unit regularly 2 times in a week; the patient received a course of ciprofloxacin twice daily for 3 days without a response. The bacterium was isolated using a MacConkey and blood agar (HiMedia, Mumbai, India), then was identified using routine conventional microbiology methods [61] and Chromogenic UTI media (bioMérieux, Lyon, France). The isolate was identified as a hypermucoviscous strain using the string test [62]. Antimicrobial susceptibility testing was performed using the disk diffusion method to test the activity of amoxicillin-clavulanate (30 µg), cefuroxime (30 µg), ceftriaxone (30 µg), ceftazidime (30 µg), cephalexin (30 µg), meropenem (10 µg), imipenem (10 µg), amikacin (30 µg), gentamicin

(10 µg), ciprofloxacin (5 µg), trimethoprim-sulfamethoxazole (25 µg), and nitrofurantoin (300 µg). *K. pneumoniae* ATCC 700603 was used for testing the quality of the culture media, antibiotic disc, and MIC. CLSI guidelines [30] were used for the susceptibility test results interpretation. DNA was extracted using the quinidine chloride protocol [63]. The gel electrophoresis and Nanodrop, Qubit (Thermo Scientific TM, Carlsbad, CA, USA), were used for the estimation of the integrity and quantification of the extracted DNA.

#### *4.2. Minimum Inhibitory Concentration (MIC)*

The microtitre broth dilution method [64] was used to determine the minimum inhibitory concentration of ciprofloxacin, gentamicin, cefotaxime, ampicillin, chloramphenicol, and tetracycline. A two-fold serial dilution of the antibiotics was prepared in Muller– Hinton (MH) broth, and 100 µL of overnight-grown bacteria adjusted to 5–10<sup>5</sup> CFU/mL was poured into each well. The antibiotics concentration used was in the range of 2 to 1024 µg/mL [65]. MIC results were interpreted according to CLSI guidelines [30].

#### *4.3. Genome Sequencing and Assembly*

Whole-genome sequencing was conducted by Novogene Company (Beijing, China) using HiSeq 2500 platform (Illumina, San Diego, CA, USA). The generated short reads (2 × 150 bp) were assembled into contigs using a de novo assembly of Velvet v. 1.2.10 [66]; then, reads with low quality and less than 200 bp were removed. The assembled sequences were submitted to GenBank under bioproject (PRJNA767482), biosample (SAMN26332310), and accession number JAKWFM000000000, and were assigned the 9KP strain. The isolate was identified using MLST 2.0 and the Pasteur MLST. The PATRIC web server and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [67] were used for genome annotation.

#### *4.4. Plasmid Assembly and Identification*

The plasmidSPAdes tool v3.15.4 [68] was used for the assembly of the putative plasmids sequences from the illumine short read, using different k-mer sizes (21, 33, and 55). The generated plasmids were further evaluated by the Plasmid Finder 2.1 tool using 95% identity and 60% coverage. Additionally, the generated plasmids were aligned using BLASTn against the plasmid sequences obtained from the plasmid database (PLSDB); then, a local database of the obtained plasmids was generated at OmicsBox v2.1, and a local blast search was used for the identification of the plasmids. A plasmid circular map was generated by the SnapGene Viewer 6.0.2 software.

#### *4.5. Identification of Antimicrobial-Resistant Genes (ARGs) and Mobile Elements*

To identify plasmid-mediated ARGs, the generated plasmids were submitted to the Resistance Gene Identifier (RGI) 5.2.1 and ResFinder 4.0 [69] databases; hits with ≥95% identity and ≥98% coverage were accepted. Furthermore, ResFinder 4.0 was used to detect chromosomal mutations conferring resistance to antibiotics; this tool contains a hit that can be flagged to indicate whether the hit is a plasmid or chromosomally mediated. Insertion sequences (IS) were identified by an IS Finder.

#### *4.6. Prediction and Comparison of Virulence Genes*

The virulence factors of the hvKP strain were screened using RAST 2.0 and the virulence factor database (VFDB) [70]. The capsule-type genes were identified using the Kleborate v2.2.0 [71] and Pathogenwatch database. The isolate (9KP) virulence profile was compared to a list of *K. pneumoniae* strains including the most closely related strains (23KE, kkp066, kkp0e6, and kkp0e7) and those found in the VFDB database which includes *K. pneumoniae* 342, MGH78578, NTUH-K2044, 1084, HS11286, KCTC 2242, and SB3432; among these strains, two (NTUH-K2044 and KCTC 2242) were hvKP [72]. SnapGene Viewer v.6.0.2 (GSL Biotech; available at snapgene.com, accessed on 20 March 2022) was used for the visualization of the virulence genes cassettes.

#### *4.7. Comparative Genomics and Phylogenetic Analysis*

The PATRIC v3.6.12 proteome comparison tool [73] was used to perform a proteinsequence-based genome comparison using bidirectional BLASTp. The OrthoVenn2 server [74] was used for protein orthologous clustering analysis. The most closely related genomes (23KE, kkp066, and kkp0e7) and the commonly used strains (*K. pneumoniae* BAA2146, HS11286, MGH78578, NTUH-K2044, NUHL24835, and PittNDM01) for *K. pneumoniae* genome comparison [31,75,76] were used as references. The phylogenetic tree was generated and visualized by the online Interactive Tree of Life (iTOL v6) tool available at Pasteur MLST. This tool generates neighbor-joining trees from concatenated nucleotide sequences; we considered all loci that contained allele sequence identifiers and cgMLST schemes for tree generation. The tree was generated against the most similar African strains of *K. pneumoniae* submitted to the Pasteur MLST database.

#### **5. Conclusions**

This study documented the presence of a rare MDR hvKP, *K. pneumoniae* 9KP, belonging to *K2* and ST14 with hypermucoviscous; it lacked the yersiniabactin system and the common regulators (*rmpA*/*rmpA2*) of the hypermucoviscous but showed the presence of other capsule regulators, such as *RcsAB* (*rcsA* and *rcsB*) and aerobactin (*iutA*), as well as the presence of salmochelin-(*iroE, iroN*) encoding genes, which are clear markers for hvKP identification.

The MIC revealed that the isolate possessed a high resistance level against ampicillin (1024 µg/mL), tetracycline (256 µg/mL), cefotaxime (128 µg/mL), and ciprofloxacin (128 µg/mL).

The isolate possessed four antimicrobial resistance plasmids (*p*KPN3-307\_type B, *p*ECW602, *p*MDR, and *p*3K157) that carried different ARGs and transposases, indicating their possible horizontal transfer and the clonal spread. The *p*ECW602 plasmid is a novel plasmid reported recently in an extensively drug-resistant (XDR) *E. coli* isolate in China [41]; here, for the first time, we reported it in a *K. pneumoniae* (9KP) isolate with high identity (99.75%).

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/antibiotics11050596/s1, Supplementary file 1: contains Tables S1–S3, representing ARGs (Table S1), virulence factors (Table S2), and numbers of the clustered and singletons proteins in the 9KP strain compared to others (Table S3). Additionally, it contains figures from Figures S1–S7 representing the string test photograph (Figure S1), a pie chart of the annotated subsystem and genes of *K. pneumoniae* 9KP (Figure S2), a circular map of the whole-genome comparison of the 9KP strain to different *K. pneumoniae* strains (Figure S3), and a map of the *K. pneumoniae* strain 9PK plasmids (Figures S4–S7)). Supplementary file 2: contains the complete data of the whole-genome comparison of the 9KP strain to different *K. pneumoniae* strains.

**Author Contributions:** H.N.A.: conceptualization, supervision, bioinformatics analysis, writing review and editing, and funding acquisition. H.S.E.: data acquisition, carried out the microbiological analysis, writing—review and editing. O.B. and I.K.: methodology, software, data curation, formal analysis, writing—review and editing, F.A.A.: methodology, software, data curation, writing—review and editing. M.S.N.: methodology, analysis, writing—review and editing. S.H.: investigations, resources, writing—review and editing. K.C.: validation, supervision, visualization, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors extend their appreciation to the Deputyship for Research & Innovation; Ministry of Education in Saudi Arabia, for funding this research work through the project number IFPRC-072-130-2020; and King Abdulaziz University DSR, Jeddah, Saudi Arabia.

**Institutional Review Board Statement:** This study was approved by the Ethics Committee of the Khartoum State Ministry of Health (REF: 2/2021).

**Informed Consent Statement:** Not applicable because we were collecting sample remnants without the patient's identifiable information.

**Data Availability Statement:** The data for this project was submitted to GenBank under the Bioproject PRJNA767482 and in the additional files.

**Acknowledgments:** The authors extend their appreciation to the Deputyship for Research & Innovation; Ministry of Education in Saudi Arabia, for funding this research work through the project number IFPRC-072-130-2020; and King Abdulaziz University DSR, Jeddah, Saudi Arabia.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Review* **Genetic Determinants of Tigecycline Resistance in** *Mycobacteroides abscessus*

**Hien Fuh Ng and Yun Fong Ngeow \***

Centre for Research on Communicable Diseases, Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Kajang 43000, Malaysia; hfng@utar.edu.my

**\*** Correspondence: ngeowyf@utar.edu.my; Tel.: +60-3-9086-0288 (ext. 158)

**Abstract:** *Mycobacteroides abscessus* (formerly *Mycobacterium abscessus*) is a clinically important, rapidgrowing non-tuberculous mycobacterium notoriously known for its multidrug-resistance phenotype. The intrinsic resistance of *M. abscessus* towards first- and second-generation tetracyclines is mainly due to the over-expression of a tetracycline-degrading enzyme known as MabTetX (*MAB\_1496c*). Tigecycline, a third-generation tetracycline, is a poor substrate for the MabTetX and does not induce the expression of this enzyme. Although tigecycline-resistant strains of *M. abscessus* have been documented in different parts of the world, their resistance determinants remain largely elusive. Recent work on tigecycline resistance or reduced susceptibility in *M. abscessus* revealed the involvement of the gene *MAB\_3508c* which encodes the transcriptional activator WhiB7, as well as mutations in the *sigH-rshA* genes which control heat shock and oxidative-stress responses. The deletion of *whiB7* has been observed to cause a 4-fold decrease in the minimum inhibitory concentration of tigecycline. In the absence of environmental stress, the SigH sigma factor (*MAB\_3543c*) interacts with and is inhibited by the anti-sigma factor RshA (*MAB\_3542c*). The disruption of the SigH-RshA interaction resulting from mutations and the subsequent up-regulation of SigH have been hypothesized to lead to tigecycline resistance in *M. abscessus*. In this review, the evidence for different genetic determinants reported to be linked to tigecycline resistance in *M. abscessus* was examined and discussed.

**Keywords:** *Mycobacteroides abscessus*; tigecycline; resistance; genetic determinants; WhiB7; SigH; RshA

#### **1. Introduction**

#### *1.1. Tigecycline*

Tigecycline is the first and only clinically available glycylcycline (a new class of tetracycline). It is a minocycline derivative, with an *N*,*N*-dimethyglycylamido moiety attached to the 90 carbon on the tetracycline four-ringed skeleton [1]. Like other tetracyclines, tigecycline is a bacteriostatic antibiotic which inhibits translation by binding to the A site of the 30S ribosomal subunit (made up of the 16S rRNA and ribosomal proteins) [2]. The protein-synthesis inhibitory activity of tigecycline is 3- and 20-fold more potent than that of minocycline and tetracycline, respectively [3]. The ability of tigecycline to escape two common mechanisms of tetracycline resistance, active efflux and ribosomal protection [2], is attributed to its bulky side chain [4]. Furthermore, a molecular modelling study demonstrated that tigecycline has additional interaction with H34 and H18 nucleotides of ribosomes, in comparison to tetracycline and minocycline [3]. These characteristics are believed to help tigecycline to bind in a different orientation and with greater affinity than tetracycline [5], thus preventing recognition by ribosomal protection proteins and Tet efflux transporters [6,7].

Tigecycline is a broad-spectrum antibiotic. It is also active against important drugresistant pathogens, such as methicillin-resistant *Staphylococcus aureus*, penicillin-resistant *Streptococcus pneumoniae*, vancomycin-resistant enterococci, and extended-spectrum betalactamase producers [2]. Furthermore, tigecycline is one of the rescue antibiotics, alongside

**Citation:** Ng, H.F.; Ngeow, Y.F. Genetic Determinants of Tigecycline Resistance in *Mycobacteroides abscessus*. *Antibiotics* **2022**, *11*, 572. https://doi.org/10.3390/ antibiotics11050572

Academic Editor: Teresa V. Nogueira

Received: 27 February 2022 Accepted: 18 April 2022 Published: 25 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

colistin, to treat infections caused by pathogens expressing the New Delhi metallo-betalactamase-1 (a carbapenemase) that confers resistance to multiple antibiotics [8]. Fastgrowing non-tuberculous mycobacteria are highly tigecycline-susceptible [9]. Specifically, this antibiotic has shown good in vitro and in vivo activities against *Mycobacteroides abscessus* complex (formerly known as *Mycobacterium abscessus* complex) [10,11]. On the other hand, slow-growing non-tuberculous mycobacteria and *Mycobacterium tuberculosis* complex are largely resistant to tigecycline [9,12].

## *1.2. The M. abscessus Complex*

*M. abscessus* complex is a species complex, consisting of *M. abscessus* subspecies *abscessus*, *M. abscessus* subspecies *massiliense* and *M. abscessus* subspecies *bolletii* (hereafter referred to as *M. abscessus*, *M. massiliense* and *M. bolletii*, respectively), that causes a wide spectrum of infections in humans, including but not limited to pulmonary and soft-tissue infections, and disseminated infections [13]. It is also one of the most important pathogens in cystic fibrosis patients [14]. More importantly, this species complex is notorious for its resistance to multiple antibiotics, mediated through its intrinsic features or through chromosomal mutations that arise under the selective pressure of antibiotic use [15]. Thus, the *M. abscessus* complex poses a major threat to clinical management and public health as treatment options for the infections caused by it are limited.

The intrinsic resistance of the *M. abscessus* complex towards first- and second-generation tetracyclines is mainly due to the over-expression of a tetracycline-degrading enzyme known as MabTetX (*MAB\_1496c*) [16]. Tigecycline is a poor substrate for the MabTetX and does not induce the expression of this enzyme [16], which could explain its potency against *M. abscessus* complex. Interestingly, tigecycline has shown synergistic activities with other antibiotics (clarithromycin, linezolid and teicoplanin) against the *M. abscessus* complex in vitro and in vivo [11,17,18]. In 2014, Wallace et al. reported that, after receiving tigecycline-containing salvage regimens for more than a month, approximately 66% of patients with *M. abscessus* complex or *M. chelonae* infections (*n* = 38) showed clinical improvement [19]. This led the authors to conclude that tigecycline might be a useful addition to other clinically available drugs in patients with these difficult-to-treat infections.

#### *1.3. Genetic Determinants of Tigecycline Resistance or Reduced Susceptibility in Other Bacteria*

Tigecycline resistance has emerged in the past 10 years and is most commonly observed among Gram-negative bacteria, mainly *Acinetobacter baumannii* and members of the Enterobacteriaceae [7]. The decreased susceptibility or resistance to tigecycline in these clinically important microorganisms has mostly been attributed to the over-expression of resistance-nodulation-cell division-type transporters, including the AcrAB efflux pumps [7]. Moreover, mutations in genes encoding the ribosomal protein S10 [20], a SAM-dependent methyltransferase [21], the acyl-sn-glycerol-3-phosphate acyltransferase [22], and proteins involved in the lipopolysaccharide core biosynthesis [23] have also been linked to tigecycline resistance in Gram-negative organisms. Another mechanism of tigecycline resistance is the TetX-mediated modification of the drug [24]. Tigecycline resistance has also been documented, albeit less frequently, in Gram-positive bacteria [7]. Through the characterization of laboratory-derived mutants, over-expression of MepA (a multidrug and toxic compound extrusion family efflux pump) and mutations in ribosomal genes (16S rRNA, ribosomal proteins and a 16S rRNA methyltransferase) were associated with resistance or decreased susceptibility to tigecycline in *S. aureus* and *S. pneumoniae*, respectively [25,26].

#### **2. Genetic Determinants of Resistance or Reduced Susceptibility to Tigecycline in** *M. abscessus*

Although tigecycline-resistant strains of *M. abscessus* complex have been documented in different parts of the world [27,28], their resistance determinants remain largely elusive. In this review, the evidence for different genetic determinants reported to be linked to tigecycline resistance or reduced tigecycline susceptibility in the subspecies *M. abscessus*

was examined and discussed. These reported genetic determinants were identified from mutants generated from *M. abscessus* ATCC 19977, the type strain of *M. abscessus*.

#### *2.1. An Intrinsic Feature Associated with Reduced Tigecycline Susceptibility: WhiB7*

In mycobacteria, WhiB7 is a transcriptional activator of intrinsic antibiotic resistance that can be induced by exposure to stresses, such as heat shock, iron deficiency and redox imbalance, and many antibiotics, including aminoglycosides, lincosamides, macrolides, pleuromutilins and tetracyclines [29–32]. In 2017, Pryjma et al. found *whiB7* (*MAB\_3508c*) to be associated with reduced tigecycline susceptibility in *M. abscessus* [33]. The deletion of the WhiB7-encoding gene caused a 4-fold decrease in the minimum inhibitory concentration (MIC—minimum inhibitory concentration) of tigecycline. Unfortunately, this group of authors did not identify the downstream effector gene(s) of WhiB7 that is linked to the reduced tigecycline susceptibility. To the best of our knowledge, this constitutes the earliest report on the genetic determinant associated with reduced tigecycline susceptibility in *M. abscessus*.

#### *2.2. Acquired Tigecycline Resistance: RshA Mutations*

In *M. abscessus,* the *sigH* gene (*MAB\_3543c*) for the sigma factor SigH and *rshA* gene (*MAB\_3542c*) for the anti-sigma factor RshA control heat shock and oxidative-stress responses. In the absence of environmental stress, RshA interacts with and inhibits SigH. In response to stress, however, the interaction between RshA and SigH is disrupted, leading to the release of SigH which would then form the RNA polymerase holoenzyme (with the core RNA polymerase) and initiate the transcription of *sigH* and other genes involved in stress response [34]. Other than heat and redox stress signals, the RshA-SigH interaction can also be disrupted by mutations in the HXXXCXXC motif of RshA [34].

Through the characterization of a tigecycline-resistant, spontaneous mutant of *M. abscessus* ATCC 19977 (MIC: 0.25 mg/L), designated as 7C (MIC: 2 mg/L), Ng et al. (2018) found the C51R mutation in the RshA to be associated with tigecycline resistance [35]. The non-species related breakpoints (sensitive ≤ 0.25 mg/L, resistant > 0.5 mg/L) proposed by the EUCAST (2018) [36] was used in this study. The C51R mutation changed the first cysteine residue in the HXXXCXXC motif to arginine. As a result, there was an up-regulation of *sigH* and other stress-response genes in 7C that was confirmed by transcriptome profiling [37]. The causal relationship between the mutation, identified by whole-genome sequencing, and the resistance phenotype was established using the complementation of 7C with the wild-type *MAB\_3542c* gene. The *whiB7* gene was not differentially expressed in 7C. In a follow-up study, Lee et al. (2021) showed that the over-expression of the *sigH* gene alone was capable of inducing tigecycline resistance in the wild-type *M. abscessus* ATCC 19977 [38]. This is supported by a recent study by Schildkraut et al. (2021) which showed an increased expression of *sigH* following an exposure of *M. abscessus* to tigecycline at a sub-inhibitory concentration, suggesting that this gene is needed for the tigecycline adaptation [39]. Although it has been well-documented that dysregulated stress response can lead to antibiotic resistance in bacteria [40], the exact mechanism or downstream gene(s) through which the RshA mutation and the *sigH* up-regulation caused a tigecycline-resistance phenotype remains unclear.

#### *2.3. SigH Mutation*

SigH is known to play two functions, which are to interact with and be inhibited by the RshA anti-sigma factor under normal circumstances and to initiate transcription in response to stressful conditions [34]. Lee et al. (2021) isolated a tigecycline-resistant mutant, designated as CL7 (MIC: 2 mg/L), which carried a stop-gain mutation (E229×) in SigH (*MAB\_3543c*) [38]. The stop-gain mutation led to a seven-amino-acid truncation in the SigH protein. Interestingly, by transforming an expression plasmid carrying the mutant *sigH* gene, the previously sensitive ATCC 19977 developed resistance towards tigecycline, suggesting that truncated SigH might retain its capability to cause tigecycline resistance.

RT-qPCR analyses of CL7 showed an over-expression of *sigH* along with stress-response genes encoding the thioredoxin and heat-shock proteins, which are the known regulon of SigH [34]. As such, these findings suggested that the SigH mutation might not be a completely loss-of-function mutation, as it only disrupted the interaction of mutated SigH with RshA but retained the SigH ability to auto-up-regulate itself and key stress genes, ultimately leading to the development of tigecycline resistance.

#### *2.4. rshA-Knockout Mutant*

The demonstration of tigecycline resistance in *M. abscessus* following the disruption of the SigH-RshA interaction and subsequent up-regulation of *sigH* led to the prediction that knocking out the *rshA* gene should also result in the development of tigecycline resistance, owing to a decreased inhibition of SigH. Unexpectedly, a recent study by Schildkraut et al. (2021) suggested otherwise [39]. Their *rshA*-knockout mutant (∆*MAB\_3542c*), derived from ATCC 19977, had neither an increase in tigecycline MIC nor a *sigH* up-regulation. A possible explanation could be that *sigH* and *rshA* are co-transcribed in a polycistronic mRNA (Figure S1A) as the genome of ATCC 19977 shows a four-bp overlap (the final four bps of the *sigH* gene are the first four bps of the *rshA* gene) (Figure S1B). As such, the deletion of *rshA* could likely result in an unwanted polar effect on the neighboring *sigH* gene. One example of such a polar effect is the introduction of synonymous mutations in the final two codons of the *sigH* gene (the alanine and stop codons) (Figure S1C). Synonymous mutations are known to alter the target gene expression [41]. In addition, the tag stop codon, introduced after the deletion of *rshA*, has been associated with a higher readthrough error rate than tga (the original stop codon) during the translation [42]. Thus, the unexpected findings by Schildkraut et al. were likely an outcome of the longer-than-usual, non-functional SigH which failed to induce tigecycline resistance and its auto-up-regulation or the altered gene expression of *sigH* due to the synonymous mutations.

#### **3. Future Perspectives and Research Areas**

Thus far, the reported genetic determinants of resistance or reduced susceptibility to tigecycline in *M. abscessus*, including WhiB7, RshA and SigH, are transcriptional regulators which respond to physiological stresses. Ribosome disruption via antibiotic exposure or mutation can lead to the production of aberrant polypeptides that are prone to oxidative modification/damage [43]. Although this aspect (tigecycline-induced oxidative damage) of tigecycline killing/inhibition has not been described before in bacteria, tigecycline has been shown to be able to induce oxidative stress in eukaryotic mitochondria [44] which have a bacterial origin [45,46]. If oxidative damage were indeed a part of the tigecycline killing/inhibition, it would be convenient for *M. abscessus* strains with WhiB7 or SigH overexpression, or RshA and SigH mutations to resist the antibiotic onslaught in clinical therapy. As oxidative damage is one of the human immune defense functions against microbes [47], and both WhiB7 and SigH are potential virulence factors in mycobacteria [48,49], it may also be interesting to investigate the pathogenicity of the WhiB7, SigH and RshA mutants in animal models.

With the emergence of tigecycline resistance in the past decade, it can be foreseen that molecular assays, such as those based on the PCR, line immunoassay and next-generation sequencing technologies, will be increasingly used for the rapid resistotyping of clinical isolates. Among the *M. abscessus* complex, studies on tigecycline resistance determinants have thus far been focused solely on *M. abscessus*. Since there is evidence suggesting a differential tigecycline susceptibility pattern among the subspecies of the *M. abscessus* complex [28], future studies in this area should focus more on the other two subspecies of *M. massiliense* and *M. bolletii*. In general, a thorough understanding of resistance determinants would help to determine the best way to utilize tigecycline for the treatment of *M. abscessus* complex infections, to prevent further escalation of tigecycline resistance in these pathogens.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antibiotics11050572/s1, Figure S1: (A) The *sigH* (*MAB\_3543c*) and *rshA* (*MAB\_3542c*) genes are transcribed as an operon. RT-PCR analysis with the forward primer annealed to the *MAB\_3543c* gene and the reverse primer annealed to the *MAB\_3542c* gene. cDNA was prepared from the RNA of ATCC 19977. NoRT: no-reverse transcription control. (B) Both genes are neighbor genes in the ATCC 19977 genome with a 4-base overlap. (C) Partial DNA sequences of *MAB\_3543c* from ATCC 19977 and ∆MAB\_3542c.

**Author Contributions:** H.F.N. and Y.F.N. conceptualized and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** Y.F.N.'s research was supported by grant 4486/000 from Universiti Tunku Abdul Rahman, Malaysia.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank Col Lin Lee and Kar Men Aw for their kind assistance in the preparation of this manuscript.

**Conflicts of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

#### **References**

