Abstract
Different samples (i.e. stool, blood, urine, sputum, wound swabs, nasal
swabs etc.) from 127 patients with suspected gram-negative infections
based on on-site performed Gram-stain from four public hospitals between
March and September 2015 in Lagos, Nigeria were analysed.Klebsiella (K .) pneumonia was identified in 43
(34%) patients. Resistance rates of these 43 strains according to the
CLSI breakpoints were as followed: cotrimoxazole (90.7%), cefuroxime
(74.4%), ofloxacin (55.8%), ceftazidime (46.5%), and cefixime (35%).
Three isolates (7%) were resistant to imipenem. All isolates were
susceptible to amoxicillin/clavulanic acid and nitrofurantoin. The
prevalence of ESBL-producing and carbapenem resistant strains (CRKP) was
69.8% and 7.0%, respectively. Of the ESBL producing isolates, twoK. pneumoniae isolates obtained from urine harbored bothbla CTX-M-1 and bla SHV and a third isolate from urine
harbored only bla CTX-M-1. Emerging of CRKP strains is an early
warning signal for the prudent use of carbapenem antibiotics in concern
to their efficacies.
Keyword: Klebsiella pneumoniae ; Imipenem; ESBL;
Emergence; Resistance; Nigeria.
Introduction
Klebsiella (K .)pneumoniae is a Gram-negative opportunistic nosocomial bacterial
pathogen. It is involved in several localized and disseminated
hospital-acquired infections such as burns infections, sepsis,
respiratory and gastrointestinal tract infections, as well as urinary
tract infections, pyogenic liver abscesses, soft tissue and wound
infection (Ranjbar et al., 2019). The emergence of
carbapenem-resistant K. pneumoniae (CRKP) strains has become an
ultimate challenge for public health globally due to their ability to
disseminate rapidly in the hospital environment and their extended
antibiotic resistance phenotypes (Rossolini, 2015). The first CRKP
isolate was reported from the USA in 2001 (Yigit et al., 2001). A few
years later, outbreaks began to appear in several countries. Nowadays,
it is the most common carbapenemase-producing Enterobacteriaceae(CPE) and is considered one of the most rapidly growing global threats
due to the high mortality in hospital-associated infections (Brink,
2019). In 2017, CRKP has been classified among those global critical
pathogens listed by WHO concerning the discovery and development of new
antibiotics.
In Nigeria, the prescription and use of carbapenem antibiotics is still
low, and there is a lack of recent information on multidrug resistance
(MDR)-associated K. pneumoniae. The present study was conducted
to investigate the prevalence of K. pneumoniae among clinical
samples of patients in four medical centers in Lagos, Nigeria, as well
as to determine the burden of ESBL and carbapenemase-producing strains.
Materials and methods
The prevalence and burden of ESBL and CRKP strains were investigated
involving 127 patients with various types of infections admitted to four
public Lagos hospitals between March and September 2015. The Central
Public Health Laboratory (CPHL), Lagos State University Teaching
Hospital (LSUTH), National Institute of Medical Research (NIMR), and Iba
Primary Health Centre (IPHC) contributed 42, 33, 32, and 20 samples,
respectively. The institutional review boards of Lagos State University
and the Nigerian Institute of Medical Research approved the study, and
the consent of the patients was sought for. In total, 37 urine samples,
30 nasal swabs, and 20 wound swabs were aseptically collected from
patients diagnosed with urinary tract infections, otitis media and
pneumonia, and soft tissue abscess. Additionally, 20 fresh feces samples
were collected from patients with gastroenteritis, and 4 ml blood was
collected from another 20 patients suffering from pyrexia of unknown
origin (PUO). All blood samples from PUO patients were subjected to the
thick-blood smear technique for the detection of the malaria parasite
(Plasmodium spp.). Bacterial culture of all samples was
done on brain heart infusion (BHI) and MacConkey agar (Oxoid, UK) and
plates were incubated at 37oC overnight under aerobic
conditions. The lactose fermenting discrete colonies were subjected to
biochemical identification according to Cown and Steel 1993 (Phillips,
1993). Colonies biochemically confirmed as Klebsiella spp. were
subjected to the MICROBACT 24E identification system (Oxoid, UK). All
confirmed K. pneumoniae strains were tested against ten
antibiotics by both, disc diffusion and microdilution methods according
to the CLSI guidelines (CLSI, 2011). The following antibiotic discs were
used: cotrimoxazole (COT) (25 µg), ciprofloxacin (CIP) (5 µg), ofloxacin
(OFL) (5 µg), gentamicin (GEN) (10 µg), cefixime (CXM) (5 µg), augmentin
(AUG) (30 µg), cefuroxime (CRX) (30 µg), ceftazidime (CAZ) (30 µg),
imipenem (IMP) (30ug), nitrofurantoin (NIT) (300 µg) (Oxoid, UK). The
diameters of the zones of inhibition were measured with a ruler and
compared with a zone-interpretation chart. The microdilution test was
performed with the same set of antibiotics for consistency (Sigma,
Deisenhofen, Germany) following the manufacturer‘s instructions. In this
study, resistance was defined for isolates exhibiting intermediate
resistance and resistance. Multidrug resistant (MDR) phenotype was
defined as resistance to three or more classes of antibiotics.
All isolates that exhibited reduced susceptibility and resistance to
third-generation cephalosporin were screened for ESBL production using
Double Disc Synergy Test Method (DDST). This was done by placing the 3GC
antibiotics i.e. ceftazidime (30 µg) and ceftriaxone (30 µg) at a
distance of 15 mm (center to center) from 30 µg augmentin, 20 µg
amoxicillin and 10 µg clavulanic acid using CLSI interpretation
guidelines (CLSI, 2011). Detection of carbapenemase enzyme activity was
done following the modified Hodge test (MHT) as described by Landman and
colleagues (Landman et al., 2005). For three ESBL gene makers, thebla CTX-M-1, bla SHV, and bla TEM PCR-based screening
was conducted by PCR (Sturenburg et al., 2004, Pai et al., 1999).
Results and discussion
The prevalence of K. pneumoniae in the clinical samples was 34 %
(n=43). K. pneumoniae was present in about half (55.8%) of urine
samples and a quarter (23.3 %) of nasal swabs. Three strains (6.98 %)
were isolated from three patients with soft tissue infection, and three
strains (6.98 %) were isolated from three cases of gastroenteritis
(Table 1). Previous epidemiological investigations have shown that MDRK. pneumoniae strains harboring ESBL genes exist in environmental
sources (Obasi et al., 2017) and are usually associated with nosocomial
infections in Nigerian hospitals (Olalekan et al., 2019, Raji et al.,
2013). However, there is still little data available on the prevalence
of K. pneumoniae in Nigerian hospitals, and the published data
are limited to regional studies and/or use of a limited number of
samples (Hertz et al., 2019). Our results are comparable to the findings
of Olalekan and colleagues, who identified K. pneumoniae in 35.4
% of samples collected from four hospitals in Lagos between 2016-2018
(Olalekan et al., 2019), and Raji and co-workers, who found thatK. pneumoniae represented 31.4 % of strains collected during an
assessment of the prevalence of drug resistance and ESBL among members
of the family Enterobacteriaceae at Lagos teaching hospital (Raji
et al., 2013).
In Nigeria, malaria is endemic. Three K. pneumoniae strains
(6.98%) were isolated from patients with concomitant Plasmodium
falciparum infection. It can be speculated that K.
pneumoniae -associated bacteraemia caused exacerbation of asymptomatic
or silent malaria. Co-infections of Enterobacteriaceae with
Plasmodium falciparum have been reported in some malaria-endemic
African countries, including Nigeria, and were associated with increased
severity (Akinyemi et al., 2007). Thus, such cases were expected also in
this study. Hence, this is the first report on K.
pneumoniae-Plasmodium spp . co-infections in patients within Lagos
hospitals in recent times.
Most of isolates were resistant to cotrimoxazole (90.7%), followed by
cefuroxime (74.4%), ofloxacin (55.8%), ceftazidime (46.5%), and
cefixime (35%). Three isolates (7%) obtained from urine showed
resistance to imipenem with a MIC>8mg/L. Resistance rates
were lowest for amoxicillin/clavulanic acid and nitrofurantoin (0.0%)
followed by gentamicin (4.7%), imipenem (7%), and ciprofloxacin
(28%). In total, 30 K. pneumoniae isolates (69.8%) were ESBL
positive, of which 27 isolates (90%) were multidrug-resistant MDR
defined by resistance against three or more classes of antibiotics.
(Table 2). In these MDR K. pneumoniae isolates eleven resistance
patterns were identified and the pattern CAZ-CRX-OFL-CIP-COT
[ceftazidime/cefuroxime/ofloxacin/ciprofloxacin/cotrimoxazole] was
the most frequent one (Table 3). Two K. pneumoniae isolates from
urine (U3 and U11) harbored both bla CTX-M-1 and bla SHV,
and one isolate (U19) harbored only bla CTX-M-1. None of the
tested strains contained the bla TEM. None of the three ESBL genes
screened for was detected in the three carbapenem-resistant isolates.
The emergence of resistance to imipenem is a new phenomenon and the
explanation is inconclusive, because this drug is rarely used and
relatively expensive. None of the three ESBL-genes was detected in any
of the three carbapenem-resistant K . pneumoniae isolates
in our study. Only three genes have been checked by PCR due to limited
resources, which is a limitation of this study.
Interestingly, the three CRKP strains were found in two different
hospitals, at close distance of 20km. The emergence of ESBL due tobla CTX and bla SHV genes in K. pneumoniae has also
been reported in other recent African studies: A high distribution of
ESBL with the dominant bla CTX-M-1 gene marker was reported in
Accra and Kumasi, Ghana (Falagas et al., 2011). A high prevalence of
ESBL-producing K. pneumoniae in clinical isolates was reported in
Côte d’Ivoire (Muller-Schulte et al., 2019). Strains harboringbla CTX-M-15, and bla SHV-134 were isolated from pig and
abattoir workers in Cameroon (Founou et al., 2018).
In conclusion, this study revealed the emergence and dissemination of
carbapenem resistant K. pnuemoniae isolates and bla CTX-M-1
and bla SHV co-harboring K. pneumoniae strains in Lagos.
This study is limited by using a PCR screening tool that could only
detect bla CTX-M-1, bla SHV and bla TEM. A
comprehensive study on the real situation on carbapenemase-producingK. pneumoniae in Lagos using advanced molecular typing tools to
assess the diversity and clonal relatedness of the strains is required
to better monitor and understand the spread of MDR K. pnuemoniaein this region.
Acknowledgment: We are grateful to the staff of the Department
of Microbiology and both the management of Lagos State University (LASU)
and the Nigerian Institute of Medical Research (NIMR) for providing
consumables and technical support respectively. Part of the abstract of
this study was presented at the American Society for Tropical Medicine
and Hygiene. It was published online in the American Journal of Tropical
Medicine and Hygiene.
Funding: LASU and NIMR supported this work through provisions
of consumables as well as through the joint efforts of the team.
Competing interests: None declared.
Authors’ contributions: KOA conceived the study and designed
the study protocol; ROA and COF carried out the literature search and
antimicrobial susceptibility testing; KOA and BAI performed molecular
analysis, organized, integrated all data and carried out the
investigation. KOA, ROA, and GW interpretation of data and drafted the
manuscript; KOA, OM, MWP, HN, and GW critically revised the manuscript
for intellectual content. All authors read and approved the final
manuscript. KOA is a guarantor of the paper.