A novel and distinct variant of the superbug Klebsiella pneumoniae called hypervirulent K. pneumoniae (hvKP) has emerged which is predicted to become a major threat in the world including Asia and Western countries. In comparison to classical K. pneumoniae, hvKP strains exhibit enhanced virulence in terms of overproduction of capsular polysaccharides and antiphagocytosis resulting in severe infections such as pyogenic liver abscess, pneumonia, endophthalmitis, osteomyelitis, and necrotizing fasciitis. Moreover, the emergence of multi and pandrug-resistant (PDR) strains renders management of hvKP infections extremely challenging due to the acquisition of extended-spectrum b-lactamases (ESBLs), carbapenemases, and recently described mcr genes. Most strains causing pyogenic infections show hypermucoviscosity when grown on agar plates and cause the generation of viscous strings exceeding 5 mm between a colony and a loop; referred to as string test. These strains are much more resistant than classic K. pneumoniae to in vitro killing by serum, to phagocytosis by neutrophils and macrophages, cause liver abscess and meningitis in mice; they have thus been referred to as hvKP strains.
The first gene identified as contributing to the hypermucoviscous phenotype in the representative strain NTUH-K2044 was magA (mucoviscosity-associated gene A), which also conferred increased virulence in mouse models. In addition to magA, several virulence factors including rmpA and various siderophore genes have been identified that characterize the hypermucoviscous hypervirulent phenotype. The prototypical hypermucoviscous K1 strain NTUH-K2044 carries two rmpA plasmid-borne genes (prmpA and prmpA2) on the same large plasmid (pK2044). Siderophores are also important virulence factors that scavenge scarce ferric iron from the environment. In K. pneumoniae, aerobactin (encoded by iutA), salmochelin (also known as catechol receptors) (encoded by iroN), and yersiniabactin are the representative siderophores. The convergence of hypervirulence and multidrug resistance (MDR) in K. pneumoniae, through both acquisition of resistance genes by hypervirulent strains and the opposite phenomena, poses a consequential public health threat.
Positive string test has been reported as a factor of hypervirulence and no study has so far been carried out to observe antimicrobial resistance (AMR) pattern among string test positive K. pneumoniae in Bangladesh. Hence, this study has been done to evaluate the association of hypervirulence with multidrug resistance (MDR) in K. pneumoniae at a tertiary care medical college hospital in Bangladesh.
Materials and Methods
A total of 350 samples (wound swab, sputum, endotracheal aspirates, pus, blood, and urine) were collected following standard protocol and processed further as required from patients admitted to Dhaka Medical College Hospital, Dhaka, Bangladesh, during 2018–2019. Written informed consent was obtained from the patients and the study was approved by the institutional ethical review committee.
Isolation and identification of the organisms from culture
Phenotypic identification of the organisms was done by observing colony morphology, hemolytic criteria, staining characteristics, pigment production (in Blood and MacConkey agar media), and biochemical tests (oxidase test, catalase tests, and other biochemical reactions after inoculation in Triple Sugar Iron, Motility Indole Urea, and Simmon’s Citrate agar media). Isolated K. pneumoniae were subcultured in Mueller–Hinton agar media and preserved at −22°C for further use.
Antimicrobial susceptibility test and determination of minimum inhibitory concentration
Susceptibility to antimicrobial agents of all isolates was done by modified Kirby-Bauer disc diffusion technique using Mueller–Hinton agar plates and zones of inhibition were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The US Food and Drug Administration identified interpretive criteria that were used for the interpretation of the zone of inhibition of tigecycline. The antimicrobial discs were used according to the standard antibiotic panel for isolated organisms. Following antibiotic discs were obtained from commercial source (Oxoid Ltd, UK)-Amoxiclav (amoxicillin 20 mg and clavulanic acid 10 mg), piperacillin-tazobactam (100/10 mg), cefepime (30 mg), cefoxitin (30 mg), ceftriaxone (30 mg), cefuroxime (30 mg), ceftazidime (30 mg), amikacin (30 mg), ciprofloxacin (5 mg), imipenem (10 mg), tigecycline (15 mg), and aztreonam (30 mg). Escherichia coli ATCC 25922 was used as control strain to assess the performance of the method. The Agar dilution method was done to determine the minimum inhibitory concentration (MIC) of colistin, tigecycline, imipenem, and fosfomycin. Double-disc synergy test was done to detect ESBL-producing strains. MDR, extensively drug resistant (XDR), and PDA strains were identified according to the Center for Disease Control and Prevention (CDC) definitions.
To detect the hypermucoid phenotype, string test was done using sterile platinum wire loop to lift a colony of K. pneumoniae grown on blood agar plate. The length of the viscous string was measured by placing a ruler straight behind it.
Polymerase chain reaction and sequencing
DNA extraction of the preserved samples was done following standard extraction protocol (boiling method). This extracted DNA was kept at −22°C and was further used for polymerase chain reaction (PCR).
Amplification in the thermal cycler was done using specific primers for virulence (magA, rmpA, rmpA2, iutA, iroN). Amplified products of PCR were detected by ultraviolet transillumination (Gel Doc, Major science, Taiwan) after electrophoresis on 1.5% agarose gel prepared with 1X TAE buffer and stained with ethidium bromide.
All data were compiled and edited meticulously by thorough checking and rechecking. All omissions and inconsistencies were corrected and were removed methodically.
The results of the study were recorded systematically. Data were analyzed and compared using Z test. All statistical analysis was performed using SPSS Statistics for Windows, version 15 (SPSS Inc., Chicago, III., USA). P = 0.05 was taken as a minimal level of statistical significance.
A total of 350 clinical samples were included in the present study of which, 220 (62.86%) samples yielded culture positivity. The distribution of total isolated and string test positive K. pneumoniae in different samples is shown in Table 1. The distribution of different bacterial species among 220 different culture-positive clinical samples is demonstrated in Table 2.
AMR pattern among total isolated (n = 44), string positive (n = 21), and string negative (n = 23) K. pneumoniae is shown in Table 3. Although higher resistance rates toward all the tested antibiotics (except for ceftriaxone, piperacillin-tazobactam, and colistin) were observed among the string test positive strains than that of string test negative isolates, only resistance proportions to amoxiclav and tigecycline were significantly higher among them (P < 0.05). Highest sensitivity was seen toward fosfomycin, followed by imipenem and least in vitro effective drug was cefuroxime, followed by ceftriaxone in this study. In Table 4, the MIC ranges of imipenem, tigecycline, colistin, and fosfomycin among resistant K. pneumoniae have been shown.
Distribution of MDR, XDR, PDR, and ESBL-producing strains of isolated K. pneumoniae in different samples is demonstrated in Table 5 along with their relative frequency among the string test positive and string test negative strains in Figure 1. Among the isolated strains, 15 (34.09%) were MDR, 10 (22.73%) were XDR, and 1 (2.27%) was PDR pathogen.
Distribution of virulence genes among isolated K. pneumoniae in different samples [Table 6] has been demonstrated, and the comparative distribution of these genes among the hypermucoid phenotypes or string positive and string negative strains is shown in Figure 2.
K. pneumoniae has been described over the past decades as an opportunistic pathogen associated with the health-care setting, wherein it is capable of causing severe illnesses. It is now considered a reservoir for AMR and virulence genes. The acquisition and dissemination of ESBL, carbapenem, and now colistin-resistance encoding genes make this pathogen an urgent threat to human health. Infections caused by the hypervirulent strains are reported to be associated with higher morbidity and mortality even with antibiotic-sensitive strains.
In this study, among 350 different clinical samples, 62.86% yielded culture-positive results. In the present study, among the culture-positive samples, 24.55% were E. coli which was the most common pathogen, followed by Pseudomonas aeruginosa (23.18%). K. pneumoniae accounted to be 20% in the current study. A study in India reported that 56.9% of samples yielded growth of bacteria. The high prevalence rate of Klebsiella among clinical patients has become a global concern in recent years since 24% and 19.72% prevalence rates were reported from northeastern and southeastern part of Bangladesh, respectively. These findings are in accordance with the present study.
In the present study, K. pneumoniae was found to be highly resistant to second-, third-, and fourth-generation cephalosporins, aztreonam, and ciprofloxacin. Previous studies in Bangladesh also reported very high resistance rates among isolated K. pneumoniae toward cefuroxime, ceftriaxone, ceftazidime, cefepime, and ciprofloxacin. The resistance pattern of isolated K. pneumoniae to imipenem (20.45%), tigecycline (20.45%), colistin (31.82%), and fosfomycin (13.67%) in the current study does not correspond to the findings of previous studies in Bangladesh. From 2016 to 2018, the highest resistance developing trend was observed in K. pneumoniae toward colistin. One systematic review in Bangladesh reported 18.8% median resistance to colistin which ranged from 0% to 21.4%. It may be due to the increased use of these drugs in clinical settings coupled with the horizontal transfer of genetic elements including resistance genes. Colistin is also being used unchecked in food, agriculture, poultry, and animal husbandry. One recent study in the US reported that repeated exposure of bacteria to sublethal doses of chlorhexidine which is used invariably in clinical settings might result in cross-resistance with cationic antimicrobials including colistin. Furthermore, colistin is often prescribed without knowing the culture and sensitivity report. Most of the microbiology laboratories in Bangladesh still use the disc diffusion method to determine colistin susceptibility patterns whereas detection of MIC is the most accurate method according to the CLSI guidelines.
The antibiogram of Klebsiella isolates studied in India showed higher resistance against cephalosporins and fluoroquinolones, whereas the highest sensitivity was observed toward imipenem. Higher coresistance to cephalosporins as well as quinolones could be because the plasmids that contain the ESBL gene also contain resistance genes for quinolones. and availability of these over-the-counter drugs enable people to consume without prescription in incomplete and inappropriate dose regimen. Overall, polymyxin resistance rates in K. pneumoniae vary among different regions, but it seems that the prevalence is increasing worldwide. Similarly, increased tigecycline resistance has been also observed in K. pneumoniae comparing previous studies in Dhaka Medical College Hospital.
ESBL-producing Gram-negative bacteria had decreased over time in Bangladesh. Several studies have been conducted and reported the rate of ESBL-producing bacteria as 30.01% in 2008, 25% in 2012, and 16.01% in 2014. In the present study, 13.64% K. pneumoniae were ESBL producers. Changes in the antibiotic prescribing trends such as reduced use of penicillin and cephalosporine along with increased use of imipenem which led to increasing imipenem resistance might be the cause of the low rate of ESBL producers among isolated K. pneumoniae in this study. Studies in India have shown that detection rates of ESBL producing K. pneumoniae vary from 9.6% to 81.1%. It could be due to a varied degree of exposure of this organism to beta-lactam antimicrobials or because of varied transferability of plasmids in the nature or due to the differences in the methodology and interpretation employed. The higher rate of ESBL producers in places might be because carbapenem susceptibility pattern or phenotypic metallo-b-lactamase production is not ruled out during screening for ESBL production. However, by definition, ESBL producers must remain sensitive to carbapenems. However, Rao et al.(2014) did not disclose these facts.
In this study, 47.73% of the isolated K. pneumoniae was string test positive. A study in India reported 31.3% string test positive strains. In Egypt, another study reported 40.71% string test positive K. pneumoniae. The dissimilarity may be due to time, source of sample collection, and geographical variation. Although hypermucoviscosity is associated with virulence, nonhypermucoviscous or string test negative K. pneumoniae isolates may also possess the virulence genes and show higher AMR. From this, it can be presumed that there are other factors such as K1 and K2 capsular serotypes; ST23 and CC23 sequence types; virulence plasmid pLVPK and KPHP1208 pathogenicity island or other virulence genes that might be responsible for higher resistance rates. In the present study, virulence genes magA and rmpA were detected in 42.86% and 52.38% of isolated string test positive strains, respectively. This finding is in agreement with the study findings in China and Egypt that reported the presence of these genes in 44.4% and 56.52% of the isolated K. pneumoniae, respectively. There are no data available regarding the prevalence of virulence genes and string test positivity in K. pneumoniae in Bangladesh or nearby Asian countries.
In the present study, 34.09%, 22.73%, and 2.27% of K. pneumoniae isolated from different clinical specimens were found MDR, XDR, and PDR, respectively. These findings coincide with a study in India which reported 30% MDR and 27.8% XDR K. pneumoniae isolated from these samples including wound swab, pus, blood, endotracheal aspirates, urine, and sputum. A study in Pakistan reported a much higher prevalence of MDR (74.41%), XDR (20.93%), and PDR (4.65%) K. pneumoniae which might be because the bacteria were isolated from burn wounds and urine only.
Increasing resistance to last-resort antibiotics such as imipenem, colistin, tigecycline, and fosfomycin with the high frequency of MDR, XDR, and PDR K. pneumoniae observed in this study is alarming. Despite significantly higher frequency of ESBL producers among string test positive strains of isolated K. pneumoniae in the present study, no significant association was found regarding MDR and hypervirulence among the two phenotypes. Further studies are required to establish the significance of colistin use in treating patients infected with hypermucoid phenotypes of K. pneumoniae. Moreover, routine surveillance of other factors responsible for hypervirulence as well as its association with AMR pattern is needed which may play an important role in public health policymaking.
Ethical permission for this study was obtained from the Institutional Review board of the corresponding institution, Dhaka Medical College.
Financial support and sponsorship
This study was financially supported by the Microbiology laboratory of Dhaka Medical College, Dhaka, Bangladesh.
Conflicts of interest
There are no conflicts of interest.
We would like to thank the department of microbiology for technical assistance.
Part of the study was presented at the 15th Asian conference on diarrheal disease and nutrition (ASCODD) held in Dhaka, Bangladesh, on 30 January 2020.
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