Abstract

Infections caused by Klebsiella sp. are increasingly becoming difficult to treat partly due to the rise in trend of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella sp. This study aimed to detect the prevalence of ESBL-producing Klebsiella sp. and SHV, TEM, CTX-M, and OXA beta-lactamase encoding genes in isolates of Klebsiella sp. from two healthcare facilities in Port Harcourt. A cross-sectional study of 146 clinical specimens was analyzed using standard bacteriological identification techniques. ESBL-producing Klebsiella sp. was identified using clinical laboratory standard institute standards. Beta-lactamase genes SHV, TEM, OXA, and CTXM were extracted and amplified. Of the 146 clinical specimens; 47 (32.2%) were from males and 99 (67.8%) females. The number of Klebsiella sp. isolated from the female samples 11 (73.3%) was more than 2-fold higher than those isolated from male samples 4 (26.7%). Eighty different bacterial isolates were obtained from which 15 (18.8%) were Klebsiella sp. while 12 (80%) of the identified Klebsiella sp. were β-lactamase producing Klebsiella sp. The prevalence of the Klebsiella sp. according to the different samples were 8 (53.3%), 1 (6.7%), 2 (13.3%), 2 (13.3%), 1 (6.7%), 1 (6.7%) were obtained from urine, endocervical, sputum, high vaginal, throat, hospital environment samples respectively. Isolates 2, 4-6, and 9-11 were positive for SHV gene (293 bp); all the Klebsiella isolates were positive for TEM gene (840 bp); isolates 4-8 showed positive bands for CTX-M gene (550 bp); and isolates 2, 3, 6, 8, and 12 showed positive bands for OXA gene (908 bp). The urine sample numbered 106, 42, and 55 exhibited complete resistance to all the antibiotics used and showed different types of ESBL genes: SHV; SHV, TEM, CTX-M and OXA; and OXA, and TEM respectively. This study shows that β-lactamase genes were differentially expressed in the various types of samples collected. Again, total resistance to beta-lactam drugs may not be completely dependent on the constitutive expression of several ESBL genes but TEM is present in all the sample that showed complete resistance.

1. Introduction

Over the past two decades, infections due to resistant Enterobacteriaceae have substantially increased and concomitantly affected morbidity and mortality as well as increased the cost of healthcare ^{1, 2, 3, 4}. The resistance of Enterobacteriaceae to antimicrobials usually occurs due to the presence of beta-lactamase enzymes which hydrolyze beta-lactam antibiotics capable of inducing resistance to penicillins, first, second, and third-generation cephalosporins, and some classes of aztreonam. The factors responsible for humans to harbor extended-spectrum beta-lactamase (ESBL) genes can either be innate or extrinsic ^{5, 6, 7}. The innate factors include natural host barriers such as the reduction in the production of gastric acid which is caused by proton-pump inhibitors or reduced microbial colonization due to extensive antimicrobial usage ⁸. Extrinsic factors include the frequency and intensity of physical contact with the bacteria due to movement to endemic areas, contact with pets, and foods containing the bacteria ^{9, 10, 11, 12}.

Enterobacteriaceae includes several gram-negative bacteria but those of clinical relevance were Escherichia coli, Klebsiella, Salmonella, and Shigella. Klebsiella sp. are non-motile, rod-shaped, gram-negative, encapsulated bacteria responsible for both nosocomial and community-acquired infections such as urinary tract infection, pneumonia, meningitis, thrombophlebitis, osteomyelitis, cholecystitis, and general bacteremia ^{13, 14}. ESBL-producing Klebsiella sp. were responsible for the majority of the multidrug-resistant gram-negative bacterial outbreak ^{15, 16}.

ESBL genes can be encoded chromosomally or through plasmid. However, majority of ESBL are plasmid-mediated especially the TEM and SHV ¹⁷. It is important to constantly explore the different Klebsiella sp. harboring ESBL genes in healthcare facilities to determine possible causes of therapy failure with cephalosporins and carbapenems to prevent and control infections.

There are six classes of beta-lactamases: inducible and constitute beta-lactamases; chromosomally mediated beta-lactamases; plasmid-mediated TEM-type beta-lactamases; broad-spectrum beta-lactamases, SHV-type beta-lactamases, metallo-beta-lactamases ¹³. The Ambler classification based on the amino acid sequence categorizes beta-lactamases into four classes: I (CTX-M, SHV, and TEM); II (IMP, SPM, and VIM), III (AmpC, FOX, and MOX); and IV (OXA) ¹⁸.

A comprehensive understanding of current healthcare prevalence studies of ESBL-producing Klebsiella sp is necessary to optimally control the infection and provide the best therapeutic management for patients in a particular region. The study aimed to detect ESBL genes such as SHV, TEM, CTX-M, and OXA in Klebsiella sp isolated from patients attending tertiary and primary hospitals in Rivers State.

2. Methods

The samples were collected from the Model Primary Health Center Rumuigbo Chest clinic and the University of Port Harcourt Teaching Hospital Obio/Akpor Local Government Area, Rivers State, Nigeria. The location of Rivers State in Nigeria co-ordinates: 40 45’ N 60 50’ E / 4.7500 N 6.8330 E. Its surrounding states were Imo, Abia, and Anambra to the North, Akwa Ibom to the East, Bayelsa to the West. In the South, it is bounded by the Atlantic Ocean.

The antimicrobial agents used in this study were purchased from Oxoid, UK. The following antimicrobial agents were used: Gentamicin (10 µg), Nalidixic acid (30 µg), Ofloxacin (10 µg), Ceftazidime (30 µg), Cefixime (30 µg), Cefuroxime (30 µg), Ciprofloxacin (10 µg), Augmentin (30 µg), Ampicillin (30 µg), Ceporex (10 µg), Cotrimoxazole (30 µg), Tetracycline (30 µg), Chloramphenicol, Cefotaxime, Nalidixic acids, and Streptomycin (30 µg).

A cross-sectional design was employed in this study with a total of 146 samples collected and examined. The samples collected were urine, endocervical, sputum, high vaginal, wound, throat, and urethral swab. These were collected from patients attending Rumuigbo model primary health centre and the University of Port Harcourt Teaching Hospital.

Samples were cultured on MacConkey agar and incubated at 37˚C for 24 hrs. Colonies that were positive to string tests were gram stained and Gram-negative colonies were further subjected to a series of biochemical tests such as oxidase, Kilger iron, citrate, indole, catalase, coagulase, and motility test.

The antimicrobial susceptibility test was performed using the Kirby Bauer agar disc diffusion method. In this method, a standardized quantity of each isolate matched with 0.5 McFarland solution was uniformly spread on Mueller Hinton agar plate. The antimicrobial disks were placed on the surface of the already inoculated agar plate and incubated at 37°C for 24 hours and examined for inhibition zones. Results were interpreted as recommended by the clinical laboratory standard institute (CLSI) ¹⁹. The following antimicrobial agents were tested: Gentamicin (10 µg), Nalidixic acid (30 µg), Ofloxacin (10 µg), Ceftazidime (30 µg), Cefixime (30 µg), Cefuroxime (30 µg), Ciprofloxacin (10 µg), Augmentin (30 µg), Ampicillin (30 µg), Ceporex (10 µg), Cotrimoxazole (30 µg), Tetracycline (30 µg), Chloramphenicol, Cefotaxime, Nalidixic acids, Streptomycin (30 µg). Zones of inhibition were measured in millimetres and sensitivity was recorded based on CLSI standard.

SHV, TEM, CTXM, and OXA were detected using PCR with the primers listed in Table 1 which correspond to the internal regions of the genes. The PCR was performed using Tag polymerase (MS Techno System, Osaka, Japan) in a thermal cycler (Takara Bio Inc, Shiga, Japan). The programming used for the amplification were: 94˚C for 2 minutes, 30 cycles of 94˚C for 30 s, 55-60˚C for 30 s and 72˚C for 1-2 minutes. PCR amplicons were resolved by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide. Beta-lactamase genes were detected by comparing the size of the amplicons against the molecular weight marker.

Statistical analyses were performed using GraphPad Prism software (version 8.03 Diego, CA). Prevalence and qualitative variables were assessed by percentage. To test for statistical differences in ESBL prevalence, the Chi square test was used. Statistical significance was defined as a P-value of less than 0.05 at a 95% confidence interval.

3. Results

Table 2 shows the prevalence of Klebsiella sp. in different samples. Out of the total number of 146 clinical samples collected, 80 different bacterial isolates were obtained of which 15 (18.8%) were Klebsiella sp. while 12 (80%) of the identified Klebsiella sp. were β-lactamase producing Klebsiella sp. The prevalence of the Klebsiella sp. according to the different samples were 8 (53.3%), 1 (6.7%), 2 (13.3%), 2 (13.3%), 1 (6.7%), 1 (6.7%) were obtained from urine, endocervical, sputum, high vaginal, throat, hospital environment samples respectively. There was no Klebsiella sp. isolates identified from both the wound and urethral samples.

Figure 1 shows the prevalence of Klebsiella sp. isolated based on gender. The number of Klebsiella sp. isolated from the female samples 11 (73.3%) was more than 2-fold higher than those isolated from male samples 4 (26.7%).

All the ESBL-producing Klebsiella sp. isolates confirmed by phenotypic methods were further analyzed by PCR amplification of the β-lactamase genes, TEM, SHV, CTX-M, and OXA. As explained in section 3.1, only 12 isolates of Klebsiella sp. produced β-lactamase. Figure 2 - Figure 5 show the PCR amplification products of the β-lactamase genes, SHV, TEM, CTX-M, and OXA respectively in the 12 isolates of Klebsiella sp. For all the figures, lanes 1-12 represent the amplification products of Klebsiella sp. isolates 1-12. In Figure 2, isolates 2, 4-6, and 9-11 were positive for the SHV gene (293 bp). In Figure 3, all the Klebsiella isolates were positive for the TEM gene (840 bp). In Figure 4, isolates 4-8 showed positive bands for the CTX-M gene (550 bp). In Figure 5, isolates 2, 3, 6, 8, and 12 showed positive bands for the OXA gene (908 bp).

Figure 6 showed the level of the different types of β-lactamase genes expressed in the different samples. Out of the eight types of specimens sampled (urine, endocervical, sputum, high vaginal, throat, urethral, wound, and hospital environment) in this study, only four (urine, high vaginal, endocervical, and environment) showed the presence of β-producing Klebsiella sp. All the four β-lactamase genes studied were shown in urine and high vaginal samples. Endocervical samples had all the genes except OXA while the environmental sample had all the β-lactamase gene except SHV.

Of the 28 PCR amplicons; U 106 showed positive amplification for SHV only, U 16 encoded for TEM only, U 30 encoded for OXA and TEM genes, U 35 encoded for SHV and TEM genes, U 42 encoded for TEM, SHV, CTX-M, and OXA, U 48 encoded for TEM, SHV and CTXM genes, U 55 encoded for TEM and OXA genes, U 56 encoded for TEM, SHV and OXA, V 17 encoded for TEM and SHV genes, V 18 encoded for TEM and CTXM gene, E 17 encoded for TEM, SHV and CTXM gene while L 16 encoded for TEM, CTXM and OXA gene (Table 3).

Out of the 15 Klebsiella isolates, 12 isolates were positive for ESBLs, with the majority of ESBL producers from urine samples of which SHV was 27.8%, TEM 38.9%, CTX-M 11.1%, OXA 22.2% followed by the vaginal samples of which SHV was 12.5%, TEM 25.0%, CTX-M 12.5% and OXA 50.0%, endocervical samples, of which SHV was 33.3%, TEM 33.3%, CTX-M 33.3% and hospital environment of which TEM was 33.3%, CTX-M 33.3% and OXA 33.3%. (Figure 6).

4. Discussion

Drug resistance in clinical isolates of Klebsiella sp. is usually attributed to different phenomena such as expression of efflux pumps, production of beta-lactamase, and horizontal acquisition of antimicrobial factors ²⁰. It has been discovered that the simultaneous occurrence of resistant enzymes in bacteria causes difficulty in the treatment of their infections ²¹. In this study, 80% of the identified Klebsiella sp. were β-lactamase producing Klebsiella sp. This was approximately two times higher than the prevalence of 39% ²² and 42.9% ²³ of ESBL-producer noted in their study. A study had suggested there was induction of β-lactamase genes in the presence of herbal drugs ²⁴. People living in Sub-Saharan African countries use a lot of spices in foods which have medicinal values like herb. This phenomenon might explain the higher level of identified β-lactamase producing Klebsiella sp. Other countries in Latin America and Europe had reported much lower prevalence of 28.7% ²⁵ and 5.6% ²⁶ respectively in β-lactamase producing Klebsiella sp.

Based on gender, the prevalence of Klebsiella sp. in females was more than twice as high as the males (Figure 1) which could be explained based on the fact that the females are more prone to infections due to their biological complexities such as childbearing, menstruation among others. However, this finding was contrasting to the higher prevalence of Klebsiella noted in males than females in a population-based study in the British of Colombia in Canada ²⁷ and Minnesota in the United States of America ²⁸. Based on the nature of samples, urine showed the highest prevalence (53.3%) of Klebsiella sp. while the lowest prevalence (6.7%) was noted in endocervical, throat, and hospital environment. On the other hand, several studies ^{29, 30, 31} have shown that the level of Klebsiella sp. was less than 6% in the throat samples. Another study also showed that wound samples (31.2%) have the highest prevalence of the bacterium followed by urine samples (26.3%). This is relatively similar to this study in the sense that urine had the highest prevalence but contrary to our study, wound did not show any presence of the bacterium. A study showed an analogous pattern to our study where the prevalence in urine was the highest followed by sputum and throat samples ³².

Klebsiella sp. are major ESBL-producer around the globe today ³³. The genes responsible for β-lactamase could be harbored chromosomally or via plasmid-borne ³⁴. The result of PCR amplification showed that there was a differential pattern of expression of the ESBL genes in the Klebsiella sp. This ranges from the expression of one to four types of the genes studied in a single isolate (Figure 6). Although in some of the isolates, there was one or two of the β-lactamase genes absent. This was noted in isolates obtained from endocervical samples which did not have OXA and environmental samples that lacked SHV genes. Besides these two samples, all the samples used in this study showed the presence of TEM, SHV, CTX-M, and OXA.

It was noted that the urine samples showed the highest number of isolates with ESBL genes (Table 2 and Table 3). A striking observation in this study was that the urine samples numbered 42, 48, and 55, as well as endocervical sample, numbered 17 exhibited complete resistance to all the antibiotics used and showed different types of ESBL genes: SHV; SHV, TEM, CTX-M, and OXA; and OXA and TEM respectively. This observation implies that for complete resistance to OFX, PEF, CPX, AU, CN, S, CEP, NA, SXT, and PN, the presence of more than one ESBL gene may be responsible. It was also observed that TEM was present in all cases of complete resistance which might point to its importance in inducing total resistance in β-lactamase-producing Klebsiella sp.

Sub-inhibitory concentrations antibiotics induce resistance to bacteria ^{35, 36}. The majority of people living in developing nations are resorting to self-medication as a means of treating infectious and non-infectious diseases. Self-medication could lead to the administration of sub-inhibitory concentration of antimicrobials or misuse of drugs which could be responsible for high level of antimicrobial resistance. This may promote drug resistance and by the time the patients go to the hospital, the microorganisms may have become more resistant.

An explanation to the high evidence of the beta-lactamase in urine samples could be due to the horizontal transfer of beta-lactamase genes from other co-habiting pathogens or micro-flora to Klebsiella sp. It had previously been shown that bacteria in the presence of other bacteria produce biofilm which can necessitate the transfer of genetic materials to other bacteria ³⁷ which is a probable means of inducing the communication of β-lactamase genes.

Acknowledgements

We are very grateful to member of the Department of Medical Laboratory Science for who contributed to the work at the proposal stage. This is a publication from a MSc project carried out in the Department of Medical Laboratory Science, Rivers State University, Port Harcourt.

Conflict of Interest

The authors declared no conflict of interest.

Compliance with Ethical Standards

Clinical samples from patients were collected after informed and written consent from patient were obtained. The project was subjected to evaluation and subsequent approval of the Ethical Review Committee of the Model Primary Health Centre Rumuigbo Chest Clinic, Rivers State, and the University of Port Harcourt Teaching Hospital, Rivers State.

References