Molecular Characteristics and Antibiotic Resistance Profiles of Klebsiella Isolates in Mthatha, Eastern Cape Province, South Africa

The increase in the incidence of extended-spectrum β-lactamase- (ESBL-) producing Klebsiella species has become a serious problem worldwide, because of their incrimination in antibiotic resistance. The objective of this study is to investigate the resistance genes responsible for ESBL-producing Klebsiella species and carbapenemase-producing Klebsiella (CRE) isolated in Mthatha and to study their epidemiology. A prospective, descriptive study of 202 nonrepetitive samples from patients was obtained from Nelson Mandela Academic Hospital. The cultured Klebsiella isolates were subjected to antimicrobial susceptibility tests and the polymerase chain reaction of blaCTX-M, blaTEM, blaSHV, blaKPC, and blaNDM genes. Overall K. pneumoniae were the majority with 169 (83.7%) species isolates, followed by K. oxytoca with 29 (14.4%), while K. ozaenae and Raoultella ornithinolytica were 2 (0.9%) each. The prevalence of ESBL production in all Klebsiella species was 117 (57.9%). ESBL-genotypic resistance is driven in Mthatha by blaSHV 121 (77.1%) followed by blaTEM 105 (66.9%) and blaCTX-M at 89 (56.7%). The most common ESBL genotype combination among the Klebsiella was blaTEM + blaSHV + blaCTX-M at 79 (50.3%). There is a steady increase in the rate of ESBL genes in the last five years.


Introduction
The genus Klebsiella belongs to the Enterobacteriaceae family and comprises Gram-negative opportunistic nonmotile pathogens with a mucoid aspect. The gastrointestinal tract serves as a reservoir and is often the latent source for infections [1]. The genus Klebsiella is classified into four species: Klebsiella pneumoniae (K. pneumoniae), Klebsiella oxytoca (K. oxytoca), Klebsiella terrigena (K. terrigena), and Klebsiella planticola (K. planticola), with K. pneumoniae consisting of three subspecies, K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, and K. pneumoniae subsp. rhinoscleromatis [2]. K. pneumoniae is an opportunistic microorganism which causes serious diseases such as septicemia, pneumonia, urinary tract infections (UTIs), chronic lung disorders, and nosocomial infections in immunocompromised patients [3]. The emergence of extended-spectrum -lactamase-(ESBL-) producing bacteria, particularly in K. pneumoniae, is now a critical concern for the development of therapies against bacterial infection [4]. These strains are resistant to extended-spectrum betalactam antibiotics, aminoglycosides, and fluoroquinolones [5]. The currently dominant ESBLs belonging to class A in the Ambler classification are TEM , SHV , and CTX-M types. The detection of the common ESBL genes such as TEM , SHV , and CTX-M by molecular methods in the ESBL-producing bacteria and their patterns of antimicrobial resistance can provide useful information about their epidemiology and can aid in developing rational antimicrobial therapy. K. pneumoniae carbapenemase (KPC) are Ambler class A plasmid-encoded enzymes that are capable of hydrolyzing all beta-lactam antibiotics, including 2 International Journal of Microbiology monobactams, extended-spectrum cephalosporins, and carbapenems. Originally described in 2001, pathogens harbouring these antibiotic resistance enzymes have been reported from the United States of America, France, China, Sweden, Norway, Colombia, and Brazil. Molecular detection of the KPC gene by polymerase chain reaction (PCR) assay provides laboratories with a means to quickly identify the presence of this important resistance determinant gene [6]. Though many studies have reported the drug resistance of K. pneumoniae and K. oxytoca worldwide [4,[7][8][9]. Epidemiology studies on ESBL-producing K. pneumoniae in Republic of South Africa (RSA) from different provinces have been reported [10][11][12][13], but little is known in the Eastern Cape Province (ECP) about the epidemiology and molecular characteristics of ESBLs. The aim of this study was to investigate the resistance mechanisms to among ESBL-producing different Klebsiella species and carbapenemase-producing Klebsiella (CRE) isolated in Mthatha and surrounding areas and to study antimicrobial susceptibility to parenteral and oral antimicrobials.  Figure 1 shows the specimen catchment area, that is, Mthatha and surrounding clinics. Mthatha (formerly Umtata) is the main town of the King Sabata Dalindyebo Local Municipality in the Oliver Reginald Tambo District of the ECP in South Africa. Study areas and health facilities in Mthatha and surrounding areas were primary health centres/clinics, secondary district hospitals, and a tertiary/academic hospital.

Specimens.
Nonduplicate, randomly selected Klebsiella isolates were collected from Mthatha and surrounding-area clinics. Specimens included blood culture and catheter tips, swabs from abscesses, eye, ear, and vagina, sputum and throat swabs, urine, and sterile fluids (plural fluid, synovial fluid, etc.). Demographic data of the patients recorded were date of specimen collection, age, gender, specimen, tests ordered, and hospital/clinic and provisional diagnosis.

Microbiologic Methods.
All samples were routinely cultured on MacConkey and blood agar plates. Blood and sputum were also cultured on chocolate agar. All suspected colonies were identified by gram staining, colony characteristics, motility, and so forth. Strains were identified to the species level with bioMérieux API20E and confirmed by Siemens MicroScan Negative ID panel Type 2. MICs were determined using MicroScan dehydrated broth microdilution panel negative MIC Type 37 (Siemens Medical Solutions Diagnostics, West Sacramento, CA), following the manufacturer's guidelines and Clinical Laboratory Standards Institute (CLSI) [15]. MICs were interpreted following CLSI guidelines, including the new clinical breakpoints published in 2010 for carbapenems [16]. ESBL detection: phenotypicthe ESBL detection was done as was recommended by the CLSI confirmatory procedure, by using cefotaxime (30 g) and ceftazidime (30 g) discs alone and in combination with clavulanic acid discs. K. pneumoniae (ATCC-700603) were used as the controls throughout the study [17]. The ESBL production was confirmed by MicroScan MIC 37 panel using combination of cefotaxime/K clavulanate (Cft/CA) and ceftazidime/K clavulanate (Caz/CA) [18].

Real-Time PCR for
CTX−M , TEM , SHV , NDM , and KPC . Real-time polymerase chain reaction (rPCR) was carried out in the LightCycler 2.0 instrument (Roche Applied Science, Germany) using LightCycler 480 Probes Master kit (Roche Diagnostics, USA). The CTX-M , TEM , and SHV for ESBL and NDM and KPC genes for CRE were amplified by singleplex rPCR using the primers shown in Table 1. Primers were designed by TIB-Molbiol (Berlin, Germany) based on primers used by Turton et al. [19]. rPCR assay was performed in a 32 carousels using 20 L capillaries with each capillary containing a total volume of 20 L including 2 L of LightCycler FastStart DNA Master HybProbe, 2 L of primers (0.5 mM for each forward and reverse), 2.4 L of MgCl 2 , 2 L of extracted DNA, and water International Journal of Microbiology 3  to make up the volume of 20 L. DNA amplification was carried out using preincubation step at 95 ∘ C for 30 s, followed by 45 cycles of amplification with denaturation at 95 ∘ C for 30 s, annealing and extension at 60 ∘ C for 1 minute without the third step, and then a single cycle of cooling. Absolute quantification was carried out using the LightCycler software 4.05.

Statistical Analysis.
Our data was entered into a database using SPSS 23 for Windows (SPSS Inc., Chicago, IL). The primary analysis involved using chi-square test in order to look for association between dependable variable (ESBL production) with covariable, for example, type of hospital/clinic, gender, and type of specimens. Significant variables were included in binary logistical regression to calculate the odds ratio and 95% confidence interval. All statistical testing was two tailed and statistical significance was defined as ≤0.05.
Comparison of ESBL and non-ESBL groups was done using Epidat 3.1 software.

Results
During the study period, a total of 202 Klebsiella species were isolated from a range of clinical specimens of patients hospitalized in various wards of NMAH in Mthatha and surrounding areas with secondary hospitals and clinics. K. pneumoniae were the majority 169 (83.7%) species isolated followed by K. oxytoca 29 (14.4%) while K. ozaenae and R. ornithinolytica were 2 (0.9%). The prevalence of ESBL production in all Klebsiella species was 122 (60.4%) while ESBLproducing K. pneumoniae were 117 (69.2%) followed by K.
In the multivariate analysis (backward logistic regression), using age group from day 1 to 5 years and tertiary level of hospitalization were found to be independent risk factors for infection due to ESBL Klebsiella species (

Discussion
The emergence of plasmid mediated ESBLs among the members of Klebsiella has increased worldwide. The incidence of  [11][12][13]. ESBL in K. pneumoniae showed rate of 62% from seven public sector hospitals in 2010 [20]. We reported 57.9% (117) ESBL producers, which is less than the above-mentioned South African provinces. In all of the above-mentioned studies ECP was not represented; this could be reason for slightly less rate of ESBLs in this area. In Africa ESBLs rate in different countries have increased from 11.7 to 77.8% among K. pneumoniae (study periods: 1999-2005 and 2010) [7,21]. Our ESBL rate of 57.9% is within this range, but it is on higher side. It was interesting to note that specific ESBLs appeared to be unique to a certain country or region. Though the prevalence of ESBLs has been recognized in various parts of the country, there is only limited data on its genotypes in this part of South Africa. High antibiotic resistance in decreasing order was observed in penicillins, cephalosporins, folate pathway inhibitors, monobactams, and aminoglycosides. Percentage of low resistance was seen in carbapenems, aminoglycosides (only amikacin), glycylcyclines (tigecycline), cephamycins (cefoxitin), quinolone (levofloxacin), phosphonic acids (fosfomycin), antipseudomonal penicillins + -lactamase inhibitor (pip/tazo), and fluoroquinolones (ciprofloxacin) which can be considered for treatment of Klebsiella species in Mthatha. Although resistance of K. pneumoniae to ceftazidime is a useful marker of presence of ESBLs, some types of ESBL-producing organisms appear susceptible to ceftazidime according to standard methods, and ceftazidime resistance may be due to mechanisms other than ESBL production. We detected 61.4% resistance to ceftazidime by MIC method while 57.9% (117) Klebsiella were ESBL producers by the CLSI confirmatory test which correlates well. Antibiotic resistance to cefotaxime, ceftazidime, and cefepime was 125 (61.9%), 124 (61.4%), and 130 (64.4%), respectively, which is almost identical. This suggests that there are multiple copies of several ESBL genes in the sample, which is supported by the genotypic results in which 66.2% specimens had two or more ESBL genes (Table 3). We observed that nearly twothirds of the Klebsiella ESBL isolates were also resistant to at least 2 useful non-lactam antibiotics used to treat UTI, such as ciprofloxacin, trimethoprim-sulfamethoxazole, and gentamicin. Similar observations have been made by other investigators [22].
In this study the most common genotype was SHV 118 (75.2%) followed by TEM 99 (63.1%) and CTX-M 89 (56.7%) as last. These genotypes were found in majority of ESBL positive Klebsiella species as compared to ESBL negative Klebsiella. Our study finding is similar to the study done from Italy in which SHV was found to be the dominant ESBL genotype and also from Spain [22,23]. ESBL genotype varies in different parts of the world; CTX-M is the most prevalent gene in high proportion of samples from different parts of the world and also Morocco [7,9]. In Africa CTX-M15 is the most prevalent gene in a high proportion of the samples, disregarding country. It is usually combined with other types of CTX-M , TEM , and SHV genes [7]. In other parts of the world, for example, Canada [8] and Turkey [24], TEM was the most common ESBL genotype. The most common ESBL genotype combination among the Klebsiella species (especially in K. pneumoniae) was combination of TEM + SHV + CTX-M which is similar to a previous report from South Africa [12]. The second most common genotype combination was the combination of TEM + SHV which is similar to the study from Japan [4]. We found increased trend of Klebsiella ESBL genotypes over 4 years; this trend is seen in studies from different parts of the world, Japan and Canada [4,8].
CRE has become an international health issue and poses a major threat to the viability of currently available antibiotics. First KPC was reported from South Africa in August 2011 from private hospital in Gauteng [25]. In our study we observed resistance to carbapenems by phenotypic test as ertapenem 3.5% while meropenem and imipenem were all susceptible. But all specimens were negative for KPC International Journal of Microbiology 5

Conclusion
This is the first report of molecular characteristics and antibiotic resistance profiles of Klebsiella isolates from clinical samples of patients in Mthatha, ECP, South Africa. The majority of Klebsiella species in our area are K. pneumoniae followed by K. oxytoca and less than 1% of K. ozaenae and R. ornithinolytica. This study reveals high ESBL rate in Klebsiella species especially in K. pneumoniae in this area. We also identified ESBL-producing three genes of TEM , SHV , and CTX-M by rPCR and our antibiotic resistance in Klebsiella species in Mthatha which is driven by combination of TEM + SHV + CTX-M and the most common genotype was SHV followed by TEM and CTX-M as last. For treatment of drug resistant Klebsiella choice of antibiotics in decreasing order is carbapenems, amikacin, tigecycline, cefoxitin, levofloxacin, pip/tazo, ciprofloxacin, and fosfomycin for UTI. Fortunately we did not detect CREforming Klebsiella in Mthatha. There is steady increase in rate of ESBL genes in the last five years; therefore continuous surveillance is essential to monitor the ESBL-producing Klebsiella in hospitals and community and also for CRE. 6 International Journal of Microbiology