Occurrence of Carbapenemases, Extended-Spectrum Beta-Lactamases and AmpCs among Beta-Lactamase-Producing Gram-Negative Bacteria from Clinical Sources in Accra, Ghana

Beta-lactamase (β-lactamase)-producing Gram-negative bacteria (GNB) are of public health concern due to their resistance to routine antimicrobials. We investigated the antimicrobial resistance and occurrence of carbapenemases, extended-spectrum β-lactamases (ESBLs) and AmpCs among GNB from clinical sources. GNB were identified using matrix-assisted laser desorption/ionization time of flight–mass spectrometry (MALDITOF-MS). Antimicrobial susceptibility testing was performed via Kirby–Bauer disk diffusion and a microscan autoSCAN system. β-lactamase genes were determined via multiplex polymerase chain reactions. Of the 181 archived GNB analyzed, Escherichia coli and Klebsiella pneumoniae constituted 46% (n = 83) and 17% (n = 30), respectively. Resistance to ampicillin (51%), third-generation cephalosporins (21%), and ertapenem (21%) was observed among the isolates, with 44% being multi-drug resistant (MDR). β-lactamase genes such as AmpCs ((blaFOX-M (64%) and blaDHA-M and blaEDC-M (27%)), ESBLs ((blaCTX-M (81%), other β-lactamase genes blaTEM (73%) and blaSHV (27%)) and carbapenemase ((blaOXA-48 (60%) and blaNDM and blaKPC (40%)) were also detected. One K. pneumoniae co-harbored AmpC (blaFOX-M and blaEBC-M) and carbapenemase (blaKPC and blaOXA-48) genes. blaOXA-48 gene was detected in one carbapenem-resistant Acinetobacter baumannii. Overall, isolates were resistant to a wide range of antimicrobials including last-line treatment options. This underpins the need for continuous surveillance for effective management of infections caused by these pathogens in our settings.


Introduction
Gram-negative bacteria (GNB) are widespread in nature and cause life-threatening infections in humans [1]. The expression of beta-lactamase (β-lactamase) enzymes such as class C cephalosporinases (AmpCs), extended-spectrum β-lactamase (ESBL) and carbapenemase in GNB has been linked to increased antibiotic resistance resulting in therapeutic failure, prolonged hospital stays, increased healthcare cost and mortality [2].
Antimicrobial resistance (AMR) observed among GNB due to the expression of β-lactamase is a global public health concern [3]. In Ghana and other parts of the world, there is over-dependence on cephalosporin beta-lactam antibiotics for the treatment of infections due to their low toxicity and high potency, which have contributed to the phenomenon
Of the thirty-five ertapenem-resistant isolates, five were positive for carbapenemase genes, and these included two K. pneumoniae, one E. coli, an A. baumannii and a Providencia vermicola (P. vermicola). Table 3 shows the phenotypic and genotypic distribution of βlactamase amongst the isolates.

Discussion
This study found high levels of β-lactamase genes among GNB. The finding that E. coli and K. pneumoniae were the predominant GNB is similar to previous studies conducted in the country [4] as well as other parts of the world [3]. These organisms have been associated with antibiotic resistance and implicated in series of infections associated with high morbidity and mortality rates [24].
The most effective antibiotic in the study was cefoxitin. Cefoxitin, a cephamycin/second-generation cephalosporin, is known to exhibit bactericidal actions against GNB and is very potent in vitro against ESBL producers [25]. All the ESBL-producing GNB in our study, except one, were susceptible to cefoxitin. It is known to be a suitable alternative for

Discussion
This study found high levels of β-lactamase genes among GNB. The finding that E. coli and K. pneumoniae were the predominant GNB is similar to previous studies conducted in the country [4] as well as other parts of the world [3]. These organisms have been associated with antibiotic resistance and implicated in series of infections associated with high morbidity and mortality rates [24].
The most effective antibiotic in the study was cefoxitin. Cefoxitin, a cephamycin/secondgeneration cephalosporin, is known to exhibit bactericidal actions against GNB and is very potent in vitro against ESBL producers [25]. All the ESBL-producing GNB in our study, except one, were susceptible to cefoxitin. It is known to be a suitable alternative for carbapenems in infection treatment [25]. A substantial proportion of the study isolates were susceptible to fluoroquinolone, contrary to other studies in the country [26] and elsewhere (Nigeria, Tanzania and Rwanda) [27][28][29]. Our findings could be an indication that such a class of antibiotic might still be relevant in the treatment of GNB-associated infections in Ghana.
The proportion (22%) of ESBL phenotypes observed among the Enterobacterales is in line with studies in the country [30] and in other parts of the world [31,32], with E. coli and K. pneumoniae as frequent ESBL producers. The most predominant ESBL gene was bla CTX-M (81%), which is the case globally [33][34][35]. On the contrary, bla TEM was the predominant gene in a recent study in Ghana [30] and Nigeria [36]. The presence of bla CTX-M represents a significant public health threat since isolates harboring this gene co-harbor extra antimicrobial resistance genes making them resistant to a wide range of antimicrobials [37]. This is particularly worrying since these drugs are part of treatment regimens for the management of infections in Ghana [8]. The co-harboring of bla CTX-M and other genes could foster the growth and dissemination pathogens with multiple resistance genes in the country [37].
The finding of greater proportions of AmpC-producing GNB being MDRs was expected since the presence of AmpC β-lactamases is accompanied with resistance to first to third-generation cephalosporins, including cephymacins. It is known that AmpC enzymes may disguise the true effect of ESBLs and their identification in a bacteria strain, thus complicating the treatment of infections caused by bacteria co-harboring both genes [38]. In this study, E. coli co-harbored AmpC and ESBL (bla TEM and bla DHA-M ) genes; likewise, K. pneumoniae co-harbored AmpC and carbapenemase genes ((bla FOX-M + bla EBC-M ) + (bla KPC + bla OXA-48 )), which has also been observed in studies in Egypt [38] and Nigeria [39].
Carbapenems are the drug of choice for the treatment of severe infections [39]. In our study, the carbapenem-tested ertapenem was one of the most effective antibiotics, and this is an assurance that this drug class is still effective. However, the bla KPC gene, which is known to hydrolyze carbapenems and was initially identified in the north-eastern part of the US, has now spread throughout the US and most of the world [40]. Our study isolated carbapenem-resistant K. pneumoniae co-harboring bla KPC and bla  . To the best of our knowledge, this is one of two studies in the country to record a dual detectable carbapenemase gene (bla KPC and bla OXA-48 ) in a single carbapenem-resistant K. pneumoniae strain [41]. The presence of bla OXA-48 and bla NDM among GNB has been reported in other studies in Ghana [42]. Furthermore, we observed that the carbapenem-resistant A. baumanii (crAb) isolated from a blood culture harbored a bla OXA-48 gene in this study. In Ghana, Monheimer and colleagues identified crAb (77%) that harbored bla OXA-23 , and bla NDM genes [43]. Carbapenem-resistant A. baumannii are among the WHO-prioritized organisms under surveillance worldwide since they are associated with life-threatening infections and the frequent carriage of multi-drug resistance [17]. Their isolation from a blood sample means that such a patient will not respond to the routinely used antimicrobials. Meanwhile, data are still limited in this part of Africa on the characteristics of this pathogen [44]. As far as we know, this is the first report of a bla OXA-48 crAb recovered from a blood culture in the country. In this study, there was one K. pneumoniae strain that co-harbored carbapenemase (bla KPC + bla OXA-48 ) and AmpC genes (bla FOX-M + bla EBC-M ). Kaizeman in Iran had a similar result, but his K. pneumoniae isolates harbored ESBL genes (bla CTX-M , bla TEM and bla SHV ) and a bla VIM carbapenemase gene [45].
The ability to harbor multiple resistance genes by GNB gives them the advantage to thrive longer in healthy individuals or the environment or cause infections in immunecompromised victims. The resistant genes identified in this study fail infection control systems and subsquently lead to dire outcomes [17]. There is the need to improve infection control measures, strengthen antimicrobial resistance surveillance and identify new treatment strategies in order to mitigate this problem.

Identification of Bacteria
This study analyzed 181 archived GNB recovered from clinical sources from April 2017 to April 2018 as a form of laboratory surveillance of antimicrobial resistance and β-lactamase production. Isolates originated from urine (n = 114), wounds (n = 38), blood (n = 21), throat swabs (n = 4), stools (n = 3), and an ear swab from humans (outpatients and inpatients) with diverse infections. The isolates were sub-cultured on blood and Mac-Conkey agar. The identification and confirmation of bacteria species was achieved using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF)-mass spectrometry (Bruker Daltonics, Bremen, Germany) using Biotyper™ system 2.0 software at the genus (log(score) 1.7-2.0)) and species (log(score) ≥ 2.0) level.

Phenotypic Screening for AmpC, ESBL and Carbapenem Resistance
Investigations for AmpC, ESBL and carbapenemases were only carried out for Enterobacterales. Other GNB were screened for carbapenemase production. Using the disk diffusion assay, the presumptive detection of AmpC was performed by observing reduced susceptibility (inhibition zone < 18 mm) to cefoxitin (30 µg). The expression of ESBLs was ascertained via the combined double-disk method using cefotaxime (30 µg) and ceftazidime (30 µg) alone and in combination with clavulanic acid (10 µg). An inhibition zone difference of ≥5 mm between the single and the clavulanic acid combination disks for cefotaxime and ceftazidime confirmed ESBL expression. Isolates resistant to ertapenem (10 µg) (inhibition zone ≤ 15 mm) were deemed possible carbapenemase producers. These isolates were subjected to a confirmatory test using the Modified Hodges Test (MHT) and the modified Carbapenem Inactivation Method (mCIM) according to the CLSI guidelines. K. pneumoniae ATCC BAA 1705 and E. coli ATCC 25922 strains were used as positive and negative controls, respectively.

Molecular Detection of Antimicrobial-Resistant Gene Markers of Beta-Lactamases
Isolates that tested positive for AmpC, ESBL, or carbapenemase phenotypes were subjected to PCR to confirm the genes encoding for AmpC (bla MOX-M , bla ACC-M , bla EBC-M , bla FOX-M , bla CIT-M and bla DHA-M ) [47], ESBLs (bla CTX-M ) [48], carbapenemases (bla KPC , bla NDM , bla VIM , bla IMP and bla OXA-48 ) [49], and other β-lactamase genes (bla TEM and bla SHV ) [48] with slight modifications. Crude DNA (10 µL) was extracted from pure overnight cultures and suspended in 200µL of molecular-grade nuclease-free water, heated for 10 min at 98 • C, and centrifuged for 5 min at 4 • C and 20,000 g, as previously suggested by Quansah [42]. The supernatant was transferred into sterile 1.5 mL Eppendorf ® tubes and used as a template for the PCR. For the PCR amplification, each reaction mix of 25 µL consisted of 12.5 uL of Green PCR Master Mix (2×) (DreamTaq, Thermo Scientific, Waltham, MA, USA), 4.5 µL of primer mix, 6 µL of molecular-grade nuclease-free water and 2 µL of crude DNA template, as previously demonstrated by Khurana et al., 2018 [49]. The primers used for PCR amplification and cycling conditions are listed in Table 4. All PCR amplicons were analyzed via horizontal gel-electrophoresis in a 2% (weight/volume) agarose gel (SeaKem ® GTG ® Agarose, Lonza, Basel, Switzerland) using Tris/Acetate/EDTA 50× concentrate buffer.

Data Analysis
Data from laboratory investigations were entered into a Microsoft Excel (version 2304) database for editing and analyses. Categorical variables were summarized with frequencies and percentages.

Conclusions
We show in this study that GNB are frequently resistant to antimicrobials and harbor genes for β-lactamases such as AmpCs, ESBLs and carbapenemases which are significant determinants of antimicrobial resistance. The finding of bla KPC and bla OXA-48 producing GNB is worrying, especially in the absence of the routine detection of these pathogens in most of the clinical microbiology laboratories in the country. The study results further support the need for efficient AMR surveillance systems at local and national levels to monitor the rise and spread of these beta-lactamase-producers and other multi-drug resistant pathogens in the country.

Limitations
Not all the classes of AmpC, ESBLs and carbapenemase genes were screened using PCR. Thus, resistant isolates harboring other relevant resistance genes were not identified. Because the genomes of the isolates were not sequenced, the specific types of TEM and SHV β-lactamase, as well as the OXA-48 identified in A. baumannii, were not determined. Informed Consent Statement: This study investigated archived isolates previously collected and de-identified and therefore obtaining an informed consent was not applicable.

Data Availability Statement:
The data used/analyzed in this study can be made available by the corresponding author on request.