Multidrug-resistant clinical K. pneumoniae ST16, ST218, and ST283 and emergence of pandrug-resistant KPC-positive ST6434/K2 lineage in Iraq

Abstract

Background and aim

The increasing incidence of Klebsiella pneumoniae infections, both in the community and in hospitals, is a huge health problem. This is due to the increasing resistance of the bacteria to antibiotics and biofilm formation, as well as the presence of a capsule. This study focuses on two main objectives: to survey the most common capsular types in local isolates for the first time in Anbar, Iraq, on molecular level and to distinguish between infectious pathogen strains using multilocus sequence typing (MLST) for more efficient epidemiological and surveillance analysis, in order to determine the source of these strains (invasive or purebred).

Methodology

Multidrug-resistant (MDR) isolates adapted to genomic extraction and molecular screening of capsular type and MLST, and then to data processing by Pasteur Institut.

Results

For the first time, one isolate was registered as a new strain in the world with ST 6434; the other strains demonstrated as preregistered with ST16, ST218, and ST283. 33% of MDR isolates belonged to the capsular K2 type.

Conclusion

The study’s findings were not aligned with the global knowledge base about the distribution of capsular type in Asia. To prevent the spread of highly resistant strains, careful monitoring of virulence determinants is necessary in addition to the observation of antibiotic resistance.

Introduction

Bacterial typing is a key part of epidemiological studies on bacterial infections [1]. Diagnostic methods have improved dramatically in recent years as a result of developments in the field of molecular biotechnology. In many cases, molecular techniques have replaced phenotypic typing methods; this is because molecular techniques are more accurate and can identify bacteria at a strain level, which is more informative than phenotypic typing [2]. Therefore, typing based on nucleic acid sequencing can be used for pathogenesis typing, which can help us understand how they develop and spread, and how to prevent them. This is because conserved genes are found in all organisms and remain stable over time [2]. The most accurate and reliable way to identify and classify organisms is to sequence specific regions of their DNA that are known to be conserved across different strains, such as multilocus sequence typing (MLST) [3].

MLST is a molecular typing technique that is used to identify and categorize bacteria. It is based on the analysis of the DNA sequences of seven housekeeping genes, which are genes that have been identified as crucial for cell function and are thus highly conserved among bacteria [4]. The housekeeping genes’ DNA sequences are amplified by PCR before being sequenced. The resulting sequences are compared to sequences from a database of known MLST types. This comparison allows the bacteria to be assigned to a specific MLST type, which provides information about their genetic lineage and potential pathogenicity [5].

The rapid emergence and spread of multidrug-resistant (MDR) K. pneumoniae strains is a serious global health concern. These strains are responsible for a significant number of hospital-acquired infections, which often have high rates of morbidity and mortality [6], due to the increasing resistance toward antibiotics, as well as the presence of a capsule which makes it difficult for drugs to enter the bacterial cell. So this study aimed the following: for the first time in Iraq/Anbar, a survey of the most prevalent capsular types in local isolates was conducted, at Ramadi City Teaching Hospitals. Additionally, in order to identify the origin of these strains (invasive or purebred), it is necessary to distinguish between infectious pathogen strains using MLST for more effective epidemiological and monitoring analysis.

Methodology

Bacterial sampling

This study was carried out at the Biotechnology Department of Anbar University between March 2022 and April 2023. Patients of all ages and genders with urinary tract infection and pneumonia were included in the clinical settings from which infection samples were drawn. Furthermore, samples were collect from urine and blood and also obtained from infections associated with catheters and surgical sites from various teaching hospitals in Anbar, Iraq.

Bacterial identification

The samples identified by traditional microbiological methods which include colonial morphology, Gram staining, biochemical tests, and the Vitek 2 Compact system were utilize for final identification of organism using a GN (Gram-negative) identification card, as per the manufacturer’s instructions (bioMérieux, France).

Antibiotics sensitivity test

For each pure isolate, the antimicrobial susceptibility test was done according to the Kirby-Bauer disk diffusion method for ten different antimicrobial agents as described in the guidelines of the CLSI Institute [7], and using an AST card by the automated Vitek 2 Compact system.

String test

An inoculation loop was used to contact colonies grown on blood agar, and then, the loop was lifted from the agar surface. When a thread-like growth or a string longer than 5 mm was observed in each strain, it was classified as hypermucoviscous strain [8, 9].

Biofilm formation

Biofilm production assay, absorption values calculated, and results were classified according to the recommendations in [6].

Some phenotypic confirmation test for extended-spectrum β-lactamases (ESBLs) and carbapenemase production

• Modified Carba NP test: Bacterial strains were cultivated onto Trypticase soy agar (TSA) at 37 °C for 18–24 h. A 10-mM calibrated loopfull of the test strain was inoculated into 500 mM Tris–HCl (20 mM, pH 7.5). The suspension was subjected to vortex homogenization. Then, 30 of the cell extract were mixed with 100 mM of phenol red (Isofar) containing 0.1 mM ZnSO4 (meropenem −) and 100 mM phenol red containing 0.1 mM ZnSO4 and 6 mg/meropenem( (meropenem +). The mixtures were incubated at 37 °C for 2 h. Tests were performed in duplicate for all isolates. The color change of the meropenem-containing vial from red to yellow or orange indicated a positive result. Three independent observers recorded the results with no discordant readings [7].

• Modified Hodge test (MHT): In order to identify ESBLs and carbapenem-producing K. pneumoniae, all β-lactam-resistant strain susceptible isolates were identified and then examined by phenotypic (MHT) according to [10]. Clover leaf result gave indicator for positive for this test.

• Blue-CARBA test: In the Blue-Carba test variant, bromothymol blue was selected as the indicator, since it includes the optimal pH range (6.0 to 7.6) for most lactamases (pH 6.8), which was a key factor for a direct colony approach. A commercially and widely available imipenem (Meropenem: 500; Arwan, France) was used as the substrate for carbapenemases. The test solution consisted of an aqueous solution of bromothymol blue at 0.04% (Merck Millipore, Germany) adjusted to pH 6.0, 0.1 mmol/L ZnSO4, and 3 mg/mL of meropenem, with a final pH of 7.0. A negative-control solution (0.04% bromothymol blue solution, pH 7.0) was prepared to control the influence of bacterial components or products in the pH of the solution. A loop of a pure bacterial culture recovered from Mueller–Hinton agar (bioMérieux, France) was directly suspended in 100 µL of both test and negative-control solutions in a 96-well microtiter plate and incubated at 37 °C with agitation (150 rpm) for 2 h. Carbapenemase activity was revealed when the test and negative-control solutions, respectively, were (i) yellow versus blue, (ii) yellow versus green, or (iii) green versus blue. Noncarbapenemase producers remained blue or green on both solutions (according to [10])

• Double disk synergy test (DDST): An amoxicillin-clavulanate disk was placed at 20 mm, to the center, of cefotaxime ceftazidime, or cefepime disks on a Mueller–Hinton agar plate and incubated at 37 °C for 18–24 h and the remaining steps were done according to [11, 12].

• Modified cephalosporin inactivation methods (mCIM): The MCIM was carried out according to the original CLSI protocol. In 2 mL TSB, a suspension was prepared by suspending a complete loop, cultivated colony of the tested strain. Following that, 30 g ceftriaxone and 30 g cefotaxime disk were immersed in the suspension and incubated at 37 °C for 4 h. Following incubation, the disk was taken from the suspension and placed on a Mueller–Hinton agar plate inoculated with Escherichia coli ATCC 25922, which was then incubated at 37 °C overnight. The absence of an inhibition zone after this process shows enzymatic hydrolysis of cephalosporins (ESBL-positive), whereas the presence of a distinct inhibition zone indicates that the tested isolate does not express ESBLS activity (ESBLS-negative) [10].

• Modified carbapenem inactivation methods (eCIM): The eCIM is a modification of the carbapenem inactivation method (CIM) that uses EDTA to chelate zinc ions. Zinc ions are essential for the activity of some metallo-β-lactamases (MBLs), a type of carbapenemase. By chelating zinc ions, EDTA can inhibit the activity of MBLs and restore the activity of carbapenems; the methods were done according to [10].

Extraction of bacterial genomic DNA

The bacterial DNA was extracted according to Promega DNA Mini Kit which was provided by Promega (USA) Company.

Molecular screening of capsular type and MLST analysis

All PCR reactions were done in Applied Bio-system 2720 thermo cyclers (USA). PCR and multiplex PCR were carried out using primers of six of common capsule gene targets with their details according to [13]. MLST primers for housekeeping genes, as described by the Pasteur Institute (https://bigsdb.pasteur.fr/klebsiella/primers-used/). In most PCR reactions, Escherichia coli 25,922 genomic DNA was used as a negative control.

The PCR programs were performed according to the Pasteur Institute with few modifications and according to [13]. The PCR products were purified using a Qiagen-Germany PCR purification kit. The purified PCR DNA fragments of MLST genes were sent directly to Macrogen Company in Korea for Sanger sequencing by using an ABI3730XL automated DNA sequencer for both forward and reverse primers.

Data processing

Allelic profiles and sequence types (STs) were designed antedate the website of the Pasteur Institute database (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html).

Statistical analysis

All statistical analyses were performed using SPSS, version 18.0 (SPSS Inc., NY, USA). Chi-square tests were used to compare the relationship of capsule type with string test and biofilm.

Ethical approval

The study was approved by Ethics Committee of the Al Anbar Medical Research University (approval number 205, December 29, 2022). All individuals have given consent to participate in the current study. In addition, there are no individuals under 16 years old.

Results and discussion

Sampling and isolation

A total of 345 specimens had been collected from clinical state, 258 (74.7%) were positive for culturing while 87 (25.3%) negatives for culture. The identification of isolates was according to colonial morphology and biochemical tests, and confirmed by a Vitek 2 Compact device as a final identification of organisms by using a GN (Gram-negative) card which showed that 70 isolates belong to K. pneumoniae. Urine was the most frequent (n = 45 (64.2%)), followed by sputum (n = 1 5 (21.4%)), blood (n = 5 (7.1%)), and catheter-related infections (n = 5 (7.1%)).The negative culture cases were attributed to the reasons that some patients were under antimicrobial chemotherapy during the culture time [14].

Antimicrobial susceptibility pattern

Based on the CLSI interpretive criteria, the resistance rate among isolates toward antibiotics tested (Table 1) was as follows: levofloxacin 42% (n = 30), ciprofloxacin 32% (n = 23), amikacin 50% (n = 35), meropenem 26% (n = 18), imipenem 57% (n = 40), ceftazidime 78% (n = 55), cefotaxime 82% (n = 58), piperacillin-tazobactam 91% (n = 64), amox-clav 97% (n = 68), ampicillin 100% (n = 70), ceftriaxone 97% (n = 68). The results showed that 68.5% (48/70) of the isolates were resistant to at least one agent of three or more antimicrobial categories, and were therefore considered to be multidrug-resistant (MDR) isolates. The current study found that K. pneumoniae has the highest resistance to third-generation cephalosporins. This is consistent with other local studies [10], and is likely due to the widespread distribution of ESBL-producing bacteria.

Table 1.

Antimicrobial susceptibility results

Antibiotics		Klebsiella pneumoniae
		Resistant		Intermediate		Sensitive
		No	%	No	%	No	%
1	Amox-clav	68	97%	1	1.5%	1	1.5%
2	Ceftriaxone	63	90%	4	6%	3	4%
3	Cefotaxime	58	82%	10	14%	2	4%
4	Ceftazidime	55	78%	10	14%	5	7%
5	Meropenem	18	26%	12	17%	40	57%
6	Amikacin	35	50%	25	35%	10	15%
7	Levofloxacin	30	42%	23	33%	17	25%
8	Imipenem	40	57%	14	20%	16	23%
9	Ciprofloxacin	23	32%	35	50%	12	18%
10	Piperacillin-tazobactam	64	91%	4	6%	2	3%

The high mortality rates associated with infections caused by carbapenemase-producing K. pneumoniae are due to the fact that these infections are difficult to treat. There are few effective treatment options available, and the bacteria can often cause severe illness [15]. The world fears that MDR or XDR isolates may turn into full resistance toward the antibiotic “Superbug” as a result of a superior resistance to antibiotics in the treatment of bacterial infections which is called pandrug-resistant (PDR) [16]. So the results of this study found that five isolates are extensively drug resistant (XDR), and three isolates are PDR. These percentages vary from one city to another within the country as reported in [10]. The spread of PDR bacteria is a major threat to global health, and it is important to take steps to prevent the development of these strains; this means that they are difficult or impossible to treat with traditional antibiotics, and can cause serious infections that are difficult to cure.

Of all the multidrug-resistant (MDR) isolates surveyed, two were found to be sensitive to all antibiotics. This is a promising finding, as it suggests that some individuals are still following unique health systems that promote antibiotic stewardship.

Phenotypic detection of carbapenemases and ESBLs

Thirty isolates of MDR were selected for screening of carbapenemase and ESBL production. Fifteen/30 (50%) of the isolates gave a positive result for the presence of the carbapenemases by using the modified Carba NP and Blue CARBA tests. The modified Hodge test was used as a phenotypic confirmatory method for both serine and metallo-β-lactamase production; therefore, 28/30 (93%) gave a positive result for this examination. The imipenem-EDTA disk method was used to phenotypically detect metallo-β-lactamases. Ten of the isolates showed an enhancement of the inhibition zone, indicating the presence of a metallo-β-lactamase. ESBLs are a groups of plasmid-mediate, diverse, complex, and rapidly evolving enzymes that have a major therapeutic challenge today in the treatment of hospitalized and community-based patients. Infections due to ESBL producers range from uncomplicated UTI to life-threatening sepsis. β-Lactamases are enzymes that have the ability to hydrolyze third-generation cephalosporins and aztreonam. In general, the organisms that produce these enzymes are resistant to other classes of antibiotics and thus limit therapeutic options. The general principle in the detecting methods of ESBL depends on enhancing the activity of spectrum cephalosporins against ESBL-producing organisms by the presence of a clavulanate substance [17]. Carbapenems are the best choice for many infections caused by ESBL-producing bacteria, but recently many cases resistant to these antibiotics have been recorded [17], as found in the current study.

The first KPC-producing K pneumoniae isolate was reported in North Carolina, USA, in 1996. KPCs are now endemic in both endemic sin several countries [10]. Enterobacteriaceae-producing KPCs have also been reported in Brazil, China, Colombia, Norway, the UK, India, Sweden, and, more recently, Italy and Finland [18].

Biofilm formation

The results showed that 40/48 (83.3%) of MDR isolates produce biofilm, but with varying degrees compared to negative control. Isolates which exhibited biofilm phenotypes were categorized as follows: 47% (n = 19) demonstrated strong biofilm production, 27% (n = 11) showed moderate biofilm production, 25% (n = 10) exhibited weak biofilm production, and the remaining are other non-biofilm producers. The high productivity of biofilm may be attributed to several factors contributing to K. pneumoniae biofilm formation such polysaccharide capsule, LPS, fimbriae, pili, iron metabolism, quorum sensing, and molecules related to siderophores [6]. The polysaccharide capsule has been shown to influence different stages of biofilm formation, including initial surface adhesion and maturation. On the other hand, the sensitivity of the “MTP” method also involves measuring even small quantities of biofilm formation; this method is considered an important method for studying the early stages of biofilm formation because it uses constant conditions and can be effective in studying many of the virulence factors that contribute to biofilm formation [19].

According to Heydari and Eftekhar’s (2014) research, isolates’ capacity to form biofilms is correlated with their ability to generate -lactamase. Isolates that produced multiple types of -lactamase had been more inclined to form robust biofilms compared to isolates that produced a single kind of enzyme, while isolates which failed to generate -lactamase enzymes were incapable to form biofilms. Biofilms are complex bacterial communities composed of one or more species encased in an extracellular matrix made of proteins, carbohydrates, and genetic material derived from the bacteria themselves as well as from the host and the bacteria cells in the biofilm are shielded from immune responses and antibiotics [6].

Molecular screening of capsules

All MDR, XDR, and PDR K. pneumoniae isolates were subjected to genomic extraction and PCR analysis and the results of multiplex PCR electrophoresis showed that capsular K2 type was the most common in local isolates, accounting for 33% (16/48), and two isolates belonged to the K57 capsule type. This result is consistent with a previous study in [20]. Based on the study’s findings, the K2 capsule type is most often correlated with invasive illness or pathogenicity in local patients, where it frequently manifests as an infection in the hospital, which may account for the bacteria’s ability to resist antimicrobial agents. The other common capsule types (K1, K5, K20, and K54) were failed to be found by PCR and that may be interpreted that these capsule types are non-common in local isolates.

The study findings contradicted the established understanding of capsular type distribution around the world because the most common serotypes vary by geographic region, with K2 being more prevalent in Europe and North America, while K1 is the most common type in Asia [21]. The increase in the frequency of K2 capsule type bacteria in Asian countries, such as Iraq, may be due to the rise in tourism or commercial activity between Asia and Europe; this could have led to the transfer of these bacteria. Identifying the capsular types of K. pneumoniae is a global goal for treating infections because it can help us develop more effective therapeutics and vaccines. Different capsular types have different properties, and some are more resistant to antibiotics than others. By understanding the capsular types of K. pneumoniae, we can develop treatments that are specifically targeted to the bacteria.

Association of capsule with MDR hypermucoviscous strains and biofilm formation

The results showed a statistically significant relationship between the presence of capsule and string test at P-value 0.0324, so most of isolates that were positive for string test have a capsular type (K2). Most of isolates that were positive to PCR amplification for capsular type demonstrate a high resistance toward antibiotics, and this may be interpreted as failure of the drug to arrive to their own target in cells because the capsule (K antigen) considered a first layer in a confrontation of drugs, so the isolates considered hyper-mucoviscosity strains were more resistance, but there are some cases that do not match to common scientific knowledge about the role of the capsule in explaining antibiotic resistance [22], so the results of some hypermucoviscous strains demonstrate that although these strains are hypermucoviscous, they were sensitive for carbapenem antibiotics; that can to express a more definitive relationship between the hypermucoviscous strains which act as a barrier to DNA uptake. This limits the horizontal gene transfer of antimicrobial resistant genes, which may be one reason why hypermucoviscous strains are less likely to harbor these genes than cKp strains [23]. The results showed a statistically significant relationship between the presence of capsule with strong biofilm formation at P-value 0.002, because they are surrounded by a capsule that consists of extracellular polysaccharide, which is one of the components of the biofilm. So that the growing coexistence of these conditions is of particular apprehension as it can lead to untreatable and invasive K. pneumoniae infection.

The presence of the capsule polysaccharide around the microbial cells protects them from the process of phagocytosis by masking the O antigens and thus hindering the detection of the microbe by the phagocytes [24]. Therefore, the presence of the capsule is the most important factor for bacterial virulence by preventing the process of phagocytosis, so the strains that have a special type of capsule are considered more virulent strains and evade the immune system [25].

Molecular screening of MLST analysis

In order to study the relatedness of K. pneumoniae strains, their genomic DNA were typed by the MLST method based on the protocol described by the Institut Pasteur MLST database. All of positive capsule type (K2) isolates under study were adapted for MLST gene screening by PCR, to ensure the presence of housekeeping genes (tonB, inf B, phoE, pgi, mdh, gapA, rpoB). The results of PCR electrophoresis show that all of target genes were successfully amplified and fully identified as MLST screening.

Sequencing has been done to identify distinct alleles, and these alleles were then numbered to obtain the allelic profiles and sequence types (STs). The results were then set and analyzed by the Pasteur Institute MLST database and Pasteur scheme. Molecular typing results showed that, out of 10 isolates that were sent for sequencing and selected for their high resistance to antimicrobial agents, one of the isolates was found to be a new strain that has never been identified before (their details are listed in Table 1 with highlight red color), and three isolates were also discovered as new strains according to identified sequences of housekeeping gene with the MLST databases in Pasteur Institute but still pending to create novel sequence types (STs) by curator of Pasteur Institute, while the other strains demonstrated as preregistered with ST16, ST218, and ST283 (Table 2).

Table 2.

Sequence types (STs) and epidemiological, clinical, and microbiological characteristics of K. pneumoniae

ID	Strain name	ST	Date of isolation	Origin	Type of infection	Sample	Antibiotic resistance	Capsule type	Biofilm product	String test
1	HSAN-1-IQ	6434	2023	Infection	UTI	Urine	XDR	K2	Strong	Negative
2	HSAN11-IQ	16	2022	colonization	UTI	Urine	MDR	K2	Moderate	Positive
3	HSAN22-IQ	16	2022	infection	Pneumonia	Sputum	XDR	K2	Strong	Positive
4	HSAN45-IQ	218	2023	infection	UTI	Urine	MDR	K2	Strong	Positive
5	HSAN55-IQ	283	2022	infection	Pneumonia	Sputum	MDR	K2	Strong	Positive
6	HSAN60-IQ	283	2022	infection	Pneumonia	Sputum	MDR	K2	Moderate	Positive
7	HSAN63-IQ	283	2022	infection	Pneumonia	Sputum	XDR	K2	Strong	Positive

ST sequence type, K2 capsular type 2, UTI unary tract infection, XDR extensively drug resistance, MDR multidrug resistance [16]

The new strain was registered with ST 6434 under name HSAN-1-IQ with accession number 55929, which is isolated from one of the patients who suffered from infection with severe inflammation of the urinary tract, where the isolate was classified as XDR. This strain demonstrates full resistance to all antimicrobial agents under study. The resistance profile of ST6434 was as follows: carbapenems: resistant, β-lactamases: positive for both serine and metallo-β-lactamases, imipenem-EDTA disk method: enhancement of inhibition zone in most isolates. This means that the strain is resistant to all carbapenem antibiotics, including imipenem and meropenem. It also produces both serine and metallo-β-lactamases, which are enzymes that can break down β-lactam antibiotics. The presence of metallo-β-lactamases is particularly concerning, as these enzymes are often associated with high levels of resistance to antibiotics. In other words, the strain is very difficult to treat with antibiotics, which treatment options may be limited to combination therapy with antibiotics that are not affected by the enzymes produced by the strain, or treatment with a last resort antibiotic. The choice of treatment will depend on the severity of the infection and the patient’s overall health such as combination therapy with antibiotics that are not affected by the β-lactamases produced by the strain, and treatment with a last resort antibiotic, such as colistin.

It is important to note that the resistance profile of a strain can change over time. This is because bacteria can acquire new resistance genes through mutation or by exchanging genes with other bacteria. It is therefore important to keep up-to-date with the latest information on the resistance profiles of bacteria.

Indiscriminate consumption of antimicrobial agents and poor health monitoring or lack of medical experience in dispensing appropriate antibiotics are all reasons that led to an increase in the virulence of bacteria towards drugs. In addition to increasing prevalence of bacteria acquiring resistance genes, other factors contribute to the spread of antibiotic resistance, and therefore these reasons combine with each other to create a high pressure environment inside the bacterial cell, which may negatively affect the preserved genetic sequences, and thus contribute to the emergence of new strains, as is the case under study.

In all the world, there are 4 isolates belonging to ST283 registered in the Pasteur database only, and all of these isolates are classified as KpII (K. pneumoniae II), while the current study found three isolates belong to this ST strain and also belong to KpII, and this strain is isolated from sputum as a nosocomial infection in “Ramadi Teaching Hospital” and those strains fully identify as close to each other because molecular typing classifies they belong to the same ST. K. pneumoniae was formerly classified as containing three phylogroups, namely KpI, KpII, and KpIII, and recent studies have suggested that the phylogroup KpII should be reclassified as a new species: K. quasipneumoniae with two subspecies, K. quasipneumoniae subsp. quasipneumoniae and K. quasipneumoniae subsp. similipneumoniae, corresponding to the two subgroups, KpII-A and KpII-B, respectively [26]. The first strain with ST283 was discovered in Africa in 2016 by Institut Pasteur of Madagascar. The typing result according to MLST of the ST283 strain under study sequenced and identified these strains as K. quasipneumoniae subsp. quasipneumoniae.

The first strain with ST16 was discovered in Paris in 1997. It had the capsular type K35 and was found in a sputum specimen from a human respiratory tract. As of now, there are more than 824 strains with ST16 that have been registered as public data in the Pasteur Center. This ST16 type is considered the most common strain in Asia but for the first time was discovered in Iraq, with capsular type K2 which isolated from UTI, which had never been seen before in the world.

Conclusion

The current study has found that the capsule K2 type had been a high frequency among MDR K. pneumoniae isolated of urinary tract infection with relation of the presence of the capsule with hypermucoviscous strains. It is not enough to monitor antibiotic resistance alone but must also carefully monitor virulence determinants to ensure that highly resistant strains do not spread. Some strains are discovered as novel data of MLST for the first time in all the world such as strain with ST6434. ST16 type is considered the most common strain in Asia but for the first time was discovered in Iraq, with capsular type K2, which had never been seen before in the world.

Data Availability

According to Pasteur Institute database.

Declarations

Conflict of interest

The authors declare no competing interests.