Polyclonal Multidrug ESBL-Producing Klebsiella pneumoniae and Emergence of Susceptible Hypervirulent Klebsiella pneumoniae ST23 Isolates in Mozambique

Globally, antibiotic-resistant Klebsiella spp. cause healthcare-associated infections with high mortality rates, and the rise of hypervirulent Klebsiella pneumoniae (hvKp) poses a significant threat to human health linked to community-acquired infections and increasing non-susceptibility. We investigated the phenotypic and genetic features of 36 Klebsiella isolates recovered from invasive infections at Hospital Central of Maputo in Mozambique during one year. The majority of the isolates displayed multidrug resistance (MDR) (29/36) to cephalosporins, gentamicin, ciprofloxacin, and trimethoprim–sulfamethoxazole but retained susceptibility to amikacin, carbapenems, and colistin. Most isolates were ESBLs-producing (28/36), predominantly carrying the blaCTX-M-15 and other beta-lactamase genes (blaSHV, blaTEM-1, and blaOXA-1). Among the 16 genomes sequenced, multiple resistance genes from different antibiotic classes were identified, with blaCTX-M-15, mostly in the ISEcp1-blaCTX-M-15-orf477 genetic environment, co-existing with blaTEM-1 and aac(3)-IIa in five isolates. Our results highlight the presence of polyclonal MDR ESBL-producing K. pneumoniae from eight sequence types (ST), mostly harbouring distinct yersiniabactin within the conjugative integrative element (ICE). Further, we identified susceptible hvKp ST23, O1-K1-type isolates carrying yersiniabactin (ybt1/ICEKp10), colibactin, salmochelin, aerobactin, and hypermucoid locus (rmpADC), associated with severe infections in humans. These findings are worrying and underline the importance of implementing surveillance strategies to avoid the risk of the emergence of the most threatening MDR hvKp.


Introduction
Several species of Klebsiella, including Klebsiella pneumoniae (Kp) and Klebsiella oxytoca, are known as opportunistic pathogens that naturally reside in the nasal passages, throat, skin, and intestinal tract of healthy individuals.However, they can also be responsible for a wide spectrum of infections, such as urinary tract, intra-abdominal, and bloodstream infections, pneumonia, and meningitis [1,2].
Alarmingly, Klebsiella have emerged worldwide as multidrug-resistant (MDR) pathogens, driven by an excessive scale of use and misuse of antibiotics, which enhance the development and spread of antimicrobial resistance genes and MDR strains.
Infections caused by these strains, particularly those producing extended-spectrum beta-lactamases (ESBLs) that confer resistance to third-generation cephalosporins and monobactams, and/or carbapenemases resulting in resistance against all β-lactams, including carbapenems, present significant challenges in terms of treatment [3,4].Such infections can lead to severe illness or even fatalities, especially in individuals with weakened immune systems.They represent a significant global public health concern and are among the leading causes of hospital-acquired infections [5].
Of particular concern is Kp's contribution to the dissemination and evolution of antimicrobial resistance between Klebsiella species and other Enterobacterales.This can occur through various mechanisms, with plasmids and mobile genetic elements playing a central role [6,7].Consequently, the implementation of appropriate infection control measures in healthcare settings becomes crucial in order to effectively curb the emergence of antimicrobial resistance resulting from Kp infections.
In addition to the classical Kp strains, a hypervirulent variant, Klebsiella pneumoniae (hvKp), has emerged.These hvKp strains are associated with community-acquired infections and are characterized by the ability to cause severe and rapidly progressive infections, even in healthy individuals [8,9].
Although hvKp strains are usually susceptible to most classes of antimicrobial agents [10], the merging of virulence and resistance determinants in hybrid lineages has been increasingly reported worldwide, further worsening the situation [12].
Mozambique is facing, as are many other countries, its share of challenges concerning Klebsiella infections.Reports of MDR ESBL-producing Kp have surfaced [13][14][15][16][17][18], and there have been documented cases of MDR ESBL-producing hvKp isolates causing severe and fatal disease, particularly in vulnerable populations, like children [19].
However, in Mozambique, the available data from the main Maputo hospitals remain limited, and molecular typing of Klebsiella isolates is rare in the country, making critical the differentiation among Klebsiella isolates for effective infection control allocation.Sequencing technologies have proven valuable in achieving this goal, as well as identifying molecular mechanisms of antibiotic resistance, virulence, and plasmid diffusion.
Due to the clinical significance and knowledge gaps in Mozambique, in this study, we characterized Klebsiella spp.isolated from blood, pus, and cerebrospinal fluid over the course of a year to provide an overview of the isolates circulating at Hospital Central of Maputo (HCM) in Mozambique, a national health unit of a quaternary level.Our analysis of the genetic features of Klebsiella isolates by polymerase chain reaction (PCR) and whole genome sequencing (WGS) has yielded valuable information that can contribute to the improvement of treatment and infection prevention and control measures.

Klebsiella Isolates and Antimicrobial Susceptibility Testing
Over a year's study, a total of 36 non-duplicate Klebsiella isolates were identified, of which 35 were Kp and 1 was K. oxytoca.These isolates were mainly obtained from patient samples in the Paediatrics ward (n = 11) and Medicine ward (n = 10), with the remaining (n = 19) originating from unspecified wards at Hospital Central of Maputo, Mozambique (Table S1b).The majority of these isolates were sourced from blood samples (n = 27), followed by seven from pus samples and two from cerebrospinal fluid (CSF).
(n = 19) originating from unspecified wards at Hospital Central of Maputo, Mozambiqu (Table S1b).The majority of these isolates were sourced from blood samples (n = 27 followed by seven from pus samples and two from cerebrospinal fluid (CSF).

Antimicrobial Resistance Determinants of Klebsiella Isolates
In the analysis of beta-lactam resistance determinants, PCR was conducted on all 3 Klebsiella isolates.Among the ESBL-producing Kp and K. oxytoca isolates, 75% (27/36) wer found to carry CTX-M-type ESBLs, with blaCTX-M-15 being the most prevalent varian present in 22 isolates.Strains carrying blaCTX-M-15 were highly resistant to antibiotic including all beta-lactams tested except carbapenems.Additionally, SHV-type beta lactamases were detected in 78% (28/36) of the isolates, and other TEM β-lactamases wer found in 67% (23/36) of them.Notably, no carbapenemase genes or AmpC genes wer detected.
Furthermore, a subset of sixteen representative Klebsiella isolates was selected fo whole genome sequencing throughout the study period, based on their phenotype o antimicrobial resistance, source of isolation, and ward affiliation (Table 1).The K. oxytoca isolate (SSM90) was an ESBL producer and displayed resistance to amoxicillin-clavulanic acid, cefotaxime, ceftazidime, gentamicin, fosfomycin, and trimethoprim-sulfamethoxazole (Table S1a).

Antimicrobial Resistance Determinants of Klebsiella Isolates
In the analysis of beta-lactam resistance determinants, PCR was conducted on all 36 Klebsiella isolates.Among the ESBL-producing Kp and K. oxytoca isolates, 75% (27/36) were found to carry CTX-M-type ESBLs, with bla CTX-M-15 being the most prevalent variant, present in 22 isolates.Strains carrying bla CTX-M-15 were highly resistant to antibiotics, including all beta-lactams tested except carbapenems.Additionally, SHV-type beta-lactamases were detected in 78% (28/36) of the isolates, and other TEM β-lactamases were found in 67% (23/36) of them.Notably, no carbapenemase genes or AmpC genes were detected.
Furthermore, a subset of sixteen representative Klebsiella isolates was selected for whole genome sequencing throughout the study period, based on their phenotype of antimicrobial resistance, source of isolation, and ward affiliation (Table 1).
The genomic analysis of Klebsiella isolates confirmed the presence of bla CTX-M-15 in 13 of them.Other genes encoding beta-lactamases and oxacillinases were also prevalent: bla TEM-1 was found in 60% (9/15) of the isolates, while bla SHV class-A β-lactamase was detected in 67% (10/15) of the isolates, with both intrinsic variants (bla SHV-1 and bla SHV-11 ) and ESBL variants (bla SHV-2 and bla SHV-187 ) observed.Additionally, bla OXA-1 was identified in 47% (7/15) of the isolates.Furthermore, in K. oxytoca, the presence of bla CTX-M-15 was associated with the coexistence of ESBL bla OXY-4-1 and the beta-lactamases bla TEM-1 and bla OXA-1 (Table 2).Regarding the genetic environment of the bla CTX-M-15 gene, it was observed that in most Kp isolates and in K. oxytoca SSM90, the ISecp1 insertion sequence from the IS1380 family was located upstream of the bla CTX-M-15 gene, forming the transposition unit ISecp1bla CTX-M-15 -orf477 (Table 2).However, in Kp isolates SSM79 and SSM85, the upstream ISEcp1 copy of the bla CTX-M-15 gene was truncated and replaced with the IS26 copy (Table 2).According to the prediction of Mlplasmids, ISecp1 transposition units were situated on plasmid contigs in all ESBL-carrying isolates, with the exception of isolate SSM73, in which ISecp1-bla CTX-M-15 -orf477 was located on a chromosomal contig size of 43,172 bp.
In some instances, antimicrobial resistance genes clustered together within the isolates.For instance, in five isolates, bla CTX-M-15 genes co-existed with bla TEM-1 and aac(3)-IIa.The presence of bla OXA-1 was consistently linked with catB3 and aac(6 )-Ib-cr in all cases.Moreover, four isolates were found to possess the combination of aadA16-dfrA7-arr3-aac(6 )Ib-cr genes together.
All Kp isolates possessed the olaquindox/quinolone AB (oqxAB) efflux pump genes mediating low to intermediate quinolone resistance.Furthermore, through ResFinder analysis, point mutations were identified in the porin genes ompK36 and ompK37, several of which were predicted to contribute to the resistance to cephalosporins and favour the selection of carbapenems resistance in ESBL-producing isolates (Table S1b).Similarly, mutations in the efflux pump regulator gene acrR were identified in all isolates, except K. oxytoca (Table S1b), and were predicted to contribute to heightened resistance to fluoroquinolone.
In addition, the tigecycline-resistant isolate (SSM58P) exhibited a premature stop codon in RamR (E78*), a repressor of RamA that acts as a positive regulator for the AcrAB efflux pump system.This genetic alteration in RamR may potentially contribute to the observed phenotypic resistance to tigecycline [20].However, all other isolates exhibited a RamR (K194*) stop codon despite having a tigecycline MIC under the EUCAST breakpoint.
The K. oxytoca isolate resistant to fosfomycin lacked the fosA gene, but it exhibited mutations compared to the fosfomycin-susceptible K. pneumoniae K68 strain in the antibiotic target and transporters (MurA, GlpT, and UhpT), including MurA/S148N_S210T, GlpT/I260_VI429L, and UhpT/V434I, which are known to confer resistance to fosfomycin in Kp [21].
There were a few discrepancies between the genotype and the phenotype for some antimicrobials: the Kp isolate (SSM52A), although it harboured aac(3)-IIa/aac(6 )-Ib-cr, was phenotypically susceptible to aminoglycoside, and ten Kp isolates phenotypically susceptible to fosfomycin contained a chromosomal fosA5 gene, suggesting that this gene could be not expressed but may increase the risk of developing resistance during treatment [22].
Of the two ST23 Kp strains, one originated from the bloodstream of a patient in the Medicine ward, while the other originated from the CSF of a paediatric patient.These isolates were associated with K1 capsular (wzi1) locus and O1/O2v2 locus and harboured the most extensive array of virulence genes (virulence score 5) considered characteristic biomarkers of hypervirulent variants.These include yersiniabactin (ybt1/ICEKp10), colibactin (clb2), salmochelin (iro1), and aerobactin (iuc1).Additionally, ST23 isolates exhibited the regulator of the mucoid phenotype rmpADC (lineage 1), and one isolate carried a truncated rmpA2 locus (Table S1c).

Discussion
Klebsiella is a significant contributor to both community-acquired and hospital-acquired infections, with an alarming rise in antimicrobial resistance.Surveillance of Klebsiella antibiotic resistance is therefore mandatory in order to adapt the antibiotic combination to local resistance patterns.
In our study, we focused on determining the phenotypic and genetic features of Klebsiella isolated from a quaternary care hospital in Maputo City, the capital of Mozambique, over a period of one year.
Our investigation has revealed worrisome findings concerning Klebsiella isolates from extraintestinal sites (mainly blood samples).The majority of these isolates were found to be ESBL-producing and exhibited multidrug resistance to several crucial antibiotics, which were, primarily, trimethoprim-sulfamethoxazole, amoxicillin/clavulanate, cephalosporins, gentamicin, and ciprofloxacin, which are commonly used to treat severe Enterobacteriaceae infections prevalent among the children in the region.
These findings raise concerns regarding the sustainability of these drugs' efficacy in treating those infections in Mozambique, forcing the use of second-line antibiotics.Klebsiella isolates from this study were susceptible to carbapenems and colistin, which are usually considered last-resort therapeutic options for treating infections when other antibiotics fail.However, these alternative options are frequently unaffordable or unavailable in the country, creating significant challenges in managing infections effectively [14].
In our study, the prevalence of ESBL-producing Klebsiella isolates is alarming due to the high rate of beta-lactamases, including CTX-M-15 and other types, such as TEM, OXA, and SHV.ESBL-producing Klebsiella isolates are spreading worldwide [23], including in Africa [24,25].In particular, in Mozambique, CTX-M-15 has been often reported in E. coli [26][27][28], in Klebsiella [14,16], and, recently, also in Enterobacter cloacae complex isolates [29].In our studied isolates, the dissemination of the bla CTX-M-15 gene could be occurring through the mobility of plasmids, with IncF being the most common type, and the mobilization element ISEcp1 or IS26 truncating ISEcp1, which have been associated with the horizontal dissemination of resistance to beta-lactams [30].Interestingly, we have also noticed the same genetic context around bla CTX-M-15 (e.g. , ISEcp1-bla CTX-M-15 -orf477), which was also observed in extraintestinal E. coli strains from the same hospital [27], suggesting a possible interspecies intra-hospital spread.
Additionally, in the SSM73 Kp isolate, we observed the integration of CTX-M-15 into the chromosome via ISEcp1.The placement of resistance traits on chromosomes has elevated the worrying scenario to a new level, allowing these clones to stably spread within clinical settings, regardless of any environmental pressures [31].We have also observed an association between the carriage of CTX-M-15, bla TEM-1 , and aac(3)-IIa genes within isolates, suggesting possible co-selection mechanisms, where the acquisition of one resistance gene may facilitate the spread of others due to the selective pressures imposed by antibiotic use.
Moreover, these isolates harboured numerous resistance genes against various antibiotic classes.This accumulation of resistance underscores the complexity of antimicrobial resistance profiles in these Klebsiella isolates, posing a significant threat to public health.Immediate attention is required to address this issue before it becomes even more challenging to manage in the future.
As expected, WGS data verified that the carbapenem-susceptible isolates did not contain any carbapenemase genes.Nevertheless, certain alterations, such as I70M, I128M, and N230G, were found in the porin OmpK37.Although these alterations were not accompanied by a reduction in carbapenem MIC, they may enable antibiotics transportation into bacterial cells, thereby potentially giving rise to carbapenem resistance [32].Therefore, the administration of carbapenems in this region should be approached with caution.Moreover, carbapenem resistance mediated by NDM in E. coli has been already documented in this hospital [28], which heightens the risk of Klebsiella isolates acquiring carbapenem resistance through NDM mechanisms.This situation emphasizes the importance of vigilant surveillance and infection control measures to address the potential spread of carbapenem resistance in clinical settings.
The typing analysis by MLST highlighted the circulation of multiple STs (ST13, ST14, ST17, ST23, ST48, ST394, ST607, ST711, ST831, and ST985) among Kp isolates at HCM, with ST14, ST48, and ST23 having a global distribution.This diversity in sequence types is in line with findings from other reports [24,25], although none of them belong to the most common epidemic clones associated with antimicrobial resistance as ST258 [23] (which is, however, relatively rare in Africa) [24].ST607 was the most represented sequence type in our study, and it has been commonly identified in Kp isolates carrying bla CTX-M-15 from Malawi, South Africa, and Tanzania, neighbouring countries of Mozambique [24,25,33,34], showing the possibility of clone transmission between these countries.
Interestingly, the yersiniabactin siderophore system was found in the majority of the MDR Kp isolates, including ST14 and ST48.In particular, acquiring yersiniabactin greatly affects the severity of Kp infection and has been linked to an increased risk of bacteraemia and sepsis in these lineages.Additionally, it seems that obtaining yersiniabactin is often the first step in acquiring additional siderophores, which enhances their ability to cause invasive infections within communities [23].
It is concerning that ST23 O1-K1-type hvKp isolates carrying multiple virulence genes have been identified for the first time in Mozambique, as these strains are rarely encountered in Africa, with limited reports from Algeria, South Africa, and Madagascar [11,35,36].The identification of hvKp strains is significant in a clinical context, as they have the potential to cause infections that necessitate prolonged antibiotic treatment and are prone to disseminate through the body.Thankfully, these hvKp are currently responsive to the majority of antibiotics.
However, as seen for MDR Kp clones, hypervirulent ST23 isolates also displayed several mutations in Omp36/37 and AcrR proteins, which underlines their potential contribution to developing resistance to cephalosporins, carbapenems, and fluoroquinolones during treatment.
Furthermore, the presence of MDR ESBL-producing Klebsiella isolates carrying rmpA and wzi-K1 genes associated with hypermucoviscosity has been previously documented at the Manhiça District Hospital [19], suggesting the need to monitor the spread of these potentially dangerous clones locally.
In Mozambique, the combination of insufficient resources for infection control and improper use of antimicrobial therapy has led to a significant issue with antibiotic resistance.The results of this investigation provide valuable insights into the genotypic and phenotypic attributes displayed by Mozambican Klebsiella isolates.In particular, the presence of MDR ESBL-producing Klebsiella spp.and ST23 hvKp isolates raises concerns regarding the emergence of highly-drug-resistant clones that have access to mobile pools of virulence and antimicrobial resistance genes.This scenario could significantly hamper effective infection management efforts in hospitals and limit available treatment strategies, making it imperative to take immediate action to address this critical problem.

Sample Collection and Bacterial Identification
Gram-negative bacteria routinely isolated from blood, pus, and cerebrospinal fluid (CSF) samples obtained from patients admitted at the Hospital Central of Maputo (HCM) between March 2017 and July 2018 were collected.This study was conducted with ethical approval provided by the National Health Bioethics Committee of Mozambique (Ref.78/CNBS/2017).
The isolation of suspected Klebsiella was carried out at the Microbiology Laboratory of HCM.Sheep blood, chocolate, MacConkey, and XLD agar plates were utilized to culture and isolate the bacteria.Preliminary identification was performed using biochemical methods at the Microbiology Laboratory in the Faculty of Medicine of Eduardo Mondlane University in Maputo, Mozambique.These isolates were later sent to the Department of Biomedical Sciences at the University of Sassari in Italy for species-level identification using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) with a MALDI Biotyper instrument (Bruker Daltonics Inc., Billerica, MA, USA) and for further investigations, including antibiotic susceptibility testing as well as whole genome sequencing (WGS) and subsequent analysis.

Whole Genome Sequencing (WGS) and Analysis
For WGS, a total of 16 Klebsiella isolates originating from different wards were selected for whole genome sequencing throughout the study period, based on their phenotype of antimicrobial resistance and source of isolation.Among these, seven were recovered from pediatric patients' samples, mostly from blood, and one from cerebrospinal fluid.Additionally, five isolates were obtained from the Medicine ward, and four isolates originated from unspecified wards.Most of the sequenced isolates were multidrug resistant and ESBL producers.

Figure 1 .
Figure 1.Antimicrobial resistance rates of K. pneumoniae isolated in Hospital Central of Maput Mozambique.

Figure 1 .
Figure 1.Antimicrobial resistance rates of K. pneumoniae isolated in Hospital Central of Maputo, Mozambique.

Table 1 .
Characteristics, antimicrobial resistance profiles, and genotyping of K. pneumoniae and K. oxytoca isolates from Central Hospital of Maputo, Mozambique.