Introduction

Multidrug resistance among clinically significant gram-negative bacteria (GNB) (Escherichia coli, Klebsiella pneumoniae, Enterobacter spp. Pseudomonas aeruginosa, and Acinetobacter baumannii) has led to increased morbidity and an estimated 40% mortality in developing countries [1, 2]. These adverse outcomes are due to treatment options being limited to expensive, often unavailable, last-line drugs such as tigecycline. Carbapenems are an important β-lactam drug class used to treat serious multidrug-resistant bacterial infections. Therefore, the global increase in carbapenem resistance (CR) has been recognized as a severe health threat [3].

CR is mediated primarily by the expression of chromosomally-encoded or plasmid-encoded carbapenemases that fall into three classes: Class A (e.g., K. pneumoniae carbapenemases (KPC), Class B (e.g., New Delhi Metallo-β-lactamase (NDM), the Verona integrin-encoded Metallo-β-lactamase (VIM), and Imipenemase (IMP), and Class D (e.g.OXA-48 and -181) [4]. Secondary mechanisms for CR are the constitutive over-production of AmpC and changes in permeability due to loss or down-regulation of porins [5].

The global spread of CR is due to the carriage of CR genes on mobile genetic elements (plasmids, transposons, and integrons). These elements also harbor genes that confer resistance against multiple antibiotic classes [6, 7]. With the increased use of carbapenems, the prediction is that GNB will evolve to accumulate multiple CR genes and mechanisms of resistance [8]. This further increases the likelihood and clinical threat of the emergence of extensively drug-resistant (XDR) GNB. Growing global diversity and distribution of CR genes, which have been described extensively [9–12], and the identification of high-risk XDR strain types (STs) associated with outbreaks, further anchors this prediction. These high-risk clones include the international clone I-III [13] of A. baumannii, ST235, ST357, and ST664 of P. aeruginosa, ST258, and ST307 of K. pneumoniae [14, 15] and ST78 of Enterobacter cloacae [16].

Carbapenem resistance is a growing problem in Africa, with a reported prevalence ranging from <1% to 60% among GNB [17–19]. Recent studies in Tanzania and Uganda have shown that this prevalence could be as high as 22.4–35% with the circulation of CR genes bla_VIM, bla_OXA-48, bla_IMP, bla_KPC, and bla_NDM-1 predominantly among K. pneumonia and P. aeruginosa [20, 21]. In Kenya, only the carbapenemase genes bla_NDM-1 in K. pneumoniae, P. aeruginosa and A. baumannii, bla_OXA-23 in A. baumannii [22–24], bla_SPM in K. pneumoniae [25], and bla_VIM-2 in P. aeruginosa [26] have been identified. These are from hospitals in three Kenyan counties: Nairobi, Kiambu, and Kilifi. With increasing carbapenem use in Kenya because of the high levels of ESBL-producing Enterobacteriaceae [27–29], a concomitant rise in CR is expected that warrants close monitoring.

This study aimed to address the limited data available on the diversity and distribution of carbapenemase and other antibiotic resistance genes and strain types of clinically relevant C-NS GNB. The focus was on five bacterial species: E. coli, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter spp. from clinical isolates in hospitals from five Kenyan counties. The research demonstrated a greater variety and distribution of carbapenemase genes in Kenya than previously recognized. It further identified the therapeutic risks associated with CR infections and identified high-risk, multidrug-resistant strains. These findings underscore the importance of targeted surveillance and control of C-NS GNB.

Materials and methods

Results

Demographic and clinical characteristics of subjects

C-NS isolates were identified from forty-eight subjects in all the five study counties. Demographic and clinical information was available for all but one subject, KPA1. The majority of the C-NS isolates were from subjects in the larger referral hospitals in Nairobi (29, 60.4%) and Kisii counties (11, 22.9%). Approximately 69% of the isolates were from male subjects, 87% were inpatient, and 64% were healthcare-associated infections (HAI) per the CDC/NHSN Surveillance Definition of Healthcare-Associated Infection [34]. Approximately 64% (30/47) of isolates were from SSTI, including ear infections, injury wounds, cellulitis, abscesses, burns, bedsores, cancer lesions, amputation sites, and surgical site infections. The remaining 36% (17/47) of the isolates were from inpatient subjects with UTIs, of which 70.5% (12/17) were catheter-associated (Table 1).

Table 1

Characteristics of subjects from whom C-NS isolates were obtained.

Isolate ID	County	Sex	Patient type	Infection type	Infection site	Infection acquisition
Acinetobacter baumannii
KAB1	Nairobi	F	Inpatient	SSTI	Leg	CAI
KAB2	Nairobi	M	Inpatient	UTI	Catheter	CAI
KAB3	Nairobi	M	Inpatient	UTI	Catheter	HAI
KAB4	Nairobi	M	Inpatient	UTI	Catheter	HAI
KAB5	Nairobi	M	Inpatient	SSTI	Catheter	HAI
KAB6	Nairobi	M	Inpatient	SSTI	Leg	unclear
KAB7	Nairobi	M	Inpatient	SSTI	Leg	HAI
KAB8	Kisumu	F	Outpatient	UTI	Catheter	HAI
KAB9	Nairobi	M	Inpatient	UTI	na	HAI
KAB10	Nairobi	F	Inpatient	SSTI	Leg	HAI
KAB11	Nairobi	F	Outpatient	SSTI	Foot	HAI
KAB12	Kericho	F	Inpatient	SSTI	Leg	CAI
KAB13	Kisii	F	Inpatient	SSTI	Leg	HAI
KAB14	Kisii	M	Inpatient	SSTI	Leg	CAI
KAB15	Kisumu	F	Inpatient	UTI	Catheter	HAI
KAB16	Kisumu	F	Inpatient	SSTI	Trunk	HAI
KAB17	Kisii	F	Inpatient	SSTI	Breast	CAI
KAB18	Kisii	M	Inpatient	SSTI	Thigh	HAI
KAB19	Kisii	M	Inpatient	SSTI	Scrotum	CAI
KAB20	Kisii	F	Inpatient	SSTI	Buttocks	CAI
KAB21	Kisii	F	Inpatient	SSTI	Breast	CAI
KAB22	Kisii	M	Outpatient	SSTI	Leg	CAI
KAB23	Kisii	M	Inpatient	SSTI	Foot	CAI
KAB24	Kisii	F	Inpatient	SSTI	Abdomen	HAI
KAB25	Kericho	M	Inpatient	SSTI	Thigh	CAI
KAB26	Kericho	M	Inpatient	SSTI	Thigh	CAI
KAB27	Kisumu	M	Inpatient	SSTI	Penis	CAI
Enterobacter cloacae
KEB1	Nairobi	M	Inpatient	UTI	Catheter	HAI
KEB2	Nairobi	M	Inpatient	SSTI	Arm	HAI
Escherichia coli
KEC1	Kisii	F	Inpatient	SSTI	Back	CAI
KEC2	Nairobi	M	Inpatient	UTI	na	HAI
KEC3	Nairobi	F	Inpatient	SSTI	na	HAI
Klebsiella pneumoniae
KKPI	Nairobi	M	Outpatient	SSTI	Leg	CAI
KKP2	Kilifi	M	Outpatient	SSTI	Leg	CAI
Pseudomonas aeruginosa
KPA1	Nairobi	na	na	na	na	na
KPA2	Nairobi	M	Inpatient	UTI	Catheter	HAI
KPA3	Nairobi	M	Inpatient	UTI	Clean-catch	CAI
KPA4	Nairobi	M	Inpatient	UTI	Catheter	HAI
KPA5	Nairobi	M	Inpatient	UTI	na	HAI
KPA6	Nairobi	M	Inpatient	UTI	Catheter	HAI
KPA7	Nairobi	M	Inpatient	SSTI	Leg wound	HAI
KPA8	Nairobi	M	Inpatient	SSTI	na	HAI
KPA9	Nairobi	M	Inpatient	SSTI	na	HAI
KPA10	Nairobi	M	Inpatient	UTI	Catheter	HAI
KPA11	Nairobi	M	Inpatient	SSTI	Buttocks	HAI
KPA12	Nairobi	M	Inpatient	UTI	na	HAI
KPA13	Nairobi	M	Inpatient	UTI	na	HAI
KPA14	Nairobi	M	Outpatient	UTI	Catheter	CAI

SSTI, skin and soft tissue; UTI, urinary tract infections; CAI, community-acquired infection; HAI, healthcare-associated infection; M, male; F, female; na, information not available.

Identity and antimicrobial susceptibility patterns of carbapenem-non-susceptible isolates

The forty-eight isolates selected based on their non-susceptibility to meropenem (Table 2) were: twenty-seven A. baumannii, fourteen P. aeruginosa, three E. coli, two E. cloacae, and two K. pneumoniae (Tables 1 and 2). All isolates were resistant to meropenem except for three A. baumannii isolates (KAB14, 15, 16). These had intermediate resistance (MIC = 8), or AES interpreted intermediate resistance (MIC = 4). All the isolates were resistant to penicillin, penicillin/β-lactamase inhibitor combination, and cephalosporin drugs tested. The only exception was a single isolate (KPA3) that was susceptible to cefepime. The isolates were most susceptible to tigecycline (66%), followed by minocycline (52.9%), tetracycline (29.4%), and levofloxacin (22.9%). All fourteen P. aeruginosa isolates, two A. baumannii (KAB4, KAB6) and one K. pneumoniae (KKP1) isolates were resistant to all antibiotics tested. Eight A. baumannii isolates (KAB1-3, 7, 9–11, 13, 25–27), three E. coli (KEC1, 2, 3), and one E. cloacae (KEB1) were resistant to all but tigecycline.

Table 2

Antimicrobial susceptibility profiles of the C-NS bacterial isolates.

Isolate ID	Antibiotic susceptibility test MICs in μg/mL (Interpretation)
	MEM	CRO	PIP	TIM	FEP	LVX	TET	MIN	TGC
Acinetobacter baumannii (n = 27)
KAB1	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	> = 16 (R)	4 (S)	1 (S)
KAB2	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	> = 16 (R)	4 (S)	1 (S)
KAB3	> = 16 (R)	> = 64 (R)	> = 128 (R)	nd	> = 64 (R)	> = 8 (R)	> = 16 (R)	4 (S)	1 (S)
KAB4	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	> = 16 (R)	> = 16 (R)	> = 8 (R)
KAB5	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	> = 16 (R)	4 (S)	2 (S)
KAB6	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	> = 16 (R)	> = 16 (R)	4 (I)
KAB7	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	> = 16 (R)	8 (I)	2 (S)
KAB8	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	0.5 (S)	> = 16 (R)	< = 1 (S)	2 (S)
KAB9	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	> = 16 (R)	8 (I)	2 (S)
KAB10	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	> = 16 (R)	8 (I)	2 (S)
KAB11	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	> = 16 (R)	8 (I)	2 (S)
KAB12	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	> = 16 (R)	2 (S)	2 (S)
KAB13	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	4 (R)	> = 16 (R)	> = 16 (R)	2 (S)
KAB14	8 (I)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	4 (R)	> = 16 (R)	< = 1 (S)	< = 0.5 (S)
KAB15	4 (I*)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	4 (R)	2 (S)	< = 1 (S)	< = 0.5 (S)
KAB16	8 (I)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	4 (R)	4 (S)	< = 1 (S)	< = 0.5 (S)
KAB17	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	< = 0.12 (S)	4 (S)	< = 1 (S)	1 (S)
KAB18	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	< = 0.12 (S)	4 (S)	< = 1 (S)	1 (S)
KAB19	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	0.25 (S)	8(I)	2 (S)	4 (I)
KAB20	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	< = 0.12 (S)	4 (S)	< = 1 (S)	< = 0.5 (S)
KAB21	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	< = 0.12 (S)	4 (S)	< = 1 (S)	< = 0.5 (S)
KAB22	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	< = 0.12 (S)	4 (S)	2 (S)	1 (S)
KAB23	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	< = 0.12 (S)	4 (S)	< = 1 (S)	1 (S)
KAB24	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	< = 0.12 (S)	4 (S)	2 (S)	< = 0.5 (S)
KAB25	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	4 (R)	> = 16 (R)	> = 16 (R)	1 (S)
KAB26	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	4 (R)	> = 16 (R)	> = 16 (R)	2 (S)
KAB27	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	4 (R)	8 (I)	> = 16 (R)	2 (S)
Enterobacter cloacae (n = 2)
KEB1	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	> = 16 (R)	> = 16 (R)	2 (S)
KEB2	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	32 (R)	0.25 (S)	4 (S)	> = 16 (R)	2 (S)
Escherichia coli (n = 3)
KEC1	8 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	> = 16 (R)	> = 16 (R)	< = 0.5 (S)
KEC2	8 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	> = 16 (R)	> = 16 (R)	< = 0.5 (S)
KEC3	8 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	> = 16 (R)	> = 16 (R)	< = 0.5 (S)
Klebsiella pneumoniae(n = 2)
KKPI	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	> = 16 (R)	> = 16 (R)	> = 8 (R)
KKP2	> = 16 (R)	> = 64 (R)	> = 128 (R)	> = 128 (R)	16 (R)	1 (S)	> = 16 (R)	4 (S)	< = 0.5 (S)
Pseudomonas aeruginosa(n = 14)
KPA1	> = 8 (R)#	nd	>64 (R) #	nd	>8 (R) #	> = 4 (R) #	nd	nd	nd
KPA2	> = 16 (R)	nd	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	nd	nd	> = 8 (R)
KPA3	> = 16 (R)	nd	64 (R)	> = 128 (R)	8 (S)	> = 8 (R)	nd	nd	> = 8 (R)
KPA4	> = 16 (R)	nd	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	nd	nd	> = 8 (R)
KPA5	> = 16 (R)	nd	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	nd	nd	> = 8 (R)
KPA6	> = 16 (R)	nd	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	nd	nd	> = 8 (R)
KPA7	> = 16 (R)	nd	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	nd	nd	> = 8 (R)
KPA8	> = 16 (R)	nd	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	nd	nd	> = 8 (R)
KPA9	> = 16 (R)	nd	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	nd	nd	> = 8 (R)
KPA10	> = 8 (R)	nd	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	nd	nd	> = 8 (R)
KPA11	> = 8 (R)	nd	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	nd	nd	> = 8 (R)
KPA12	> = 16 (R)	nd	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	nd	nd	> = 8 (R)
KPA13	> = 16 (R)	nd	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	nd	nd	> = 8 (R)
KPA14	> = 8 (R)	nd	> = 128 (R)	> = 128 (R)	> = 64 (R)	> = 8 (R)	nd	nd	> = 8 (R)
No. tested	48	34	48	46	48	48	34	34	47
No. susceptible (S)	0	0	0	0	1	11	10	18	31
% S	0.0	0.0	0.0	0	2.1	22.9	29.4	52.9	66.0

MIC values that indicate non-susceptibility to the antibiotic are shaded grey.

The %S is the number of susceptible isolates as a percentage of all isolates tested for each antibiotic.

MIC, minimum inhibitory concentration; CRO, ceftriaxone; FEP, cefepime; TIM, ticarcillin-clavulanic acid; PIP, piperacillin; MEM, meropenem; LVX, levofloxacin; TET, tetracycline; TGC, tigecycline; MIN, minocycline; nd, antibiotics not tested.

*Non-susceptibility inferred by the VITEK 2^® AES system.

^# AST performed on a Microscan platform.

Carbapenemase genes detected across bacterial species

Eleven different carbapenemases from the class B Metallo-β-lactamases (bla_NDM-1, bla_NDM-5, bla_VIM-2, and bla_VIM-6) and the Class D oxacillinases (bla_OXA-23, bla_OXA-50, bla_OXA-58, bla_OXA-181, bla_OXA-66, bla_OXA-69, bla_OXA-91) were detected in the 48 isolates (Tables 3 and 4).

Table 3

The diversity of carbapenemase genes identified among the bacterial species.

Carbapenemase Ambler Class	B: Metallo- β lactamases				Class D: Oxacillinases
Bacterial species	NDM-1	NDM-5	VIM-1	VIM-6	OXA-23	OXA-58	OXA-66	OXA-69	OXA-91	OXA-181	OXA-50
A. baumannii	7				23	1	11	13	2
E. cloacae	2
P. aeruginosa	11		1	2							1
K. pneumoniae	1									1
E. coli		3
No of occurrences	21	3	1	2	23	1	11	13	2	1	1
(n = 79) (%)	(26.6)	(3.8)	(1.3)	(2.5)	(29.1)	(1.3)	(13.9)	(16.5)	(2.5)	(1.3)	(1.3)

NDM, New Delhi Metallo-β-lactamase; OXA, oxacillinase; VIM, Verona integron-encoded Metallo-β-lactamase.

Table 4

Distribution of drug resistance genes among the C-NS species and strain types.

Isolate IDs	Species (No. of isolates)	ST	Carbapenemases	Penicillinases and β-lactamases *	Aminoglycoside resistance genes #	Efflux genes	Other significant antibiotic resistance genes
KAB18-20	AB (3)	1	OXA-23, 69	ADC-25			sul2
KAB17,21–24	AB (5)	1	OXA-23, 69	ADC-25	ant(2’’)-Ia		sul2
KAB25, 26,27	AB (3)	1	NDM-1, OXA-23, 69	ADC-191	aac(3)-I, aadA1, adeC aph(3’)-Ia, aph(3’’)-Ib, aph(3’)-Via, aph(6)-Id ant(3’’)-Iia	amvA	ble, dfrA1, sat2, sul1/2, tetB
KAB16	AB (1)	1	NDM-1, OXA-69	ADC-25, CARB-16	aadA1, aph(3’)-Ia, aph(3’)-VI		mphE, msrE, sul2, dfrA1
KAB1-5,7,9, 10	AB (8)	2	OXA-23, OXA-66	ADC-25, TEM-1D	armA, aph(3’’)-Ib, aph(3’)-Ia, aph(6)-Id		mphE, msrE, tetB, sul2
KAB6,11	AB (2)	2	OXA-23, OXA-66	ADC-25, TEM-1D	armA aph(3’’)-Ib, aph(3’)-Ia, aph(6)-Id		mphE, msrE, tetB
KAB12	AB (1)	2	OXA-23, OXA-66	ADC-25, TEM-1D	aac(3)-Ia, aadA1,aph(3’)-Ia		mphE, msrE, sul1/2
KAB13	AB (1)	1475	NDM-1, OXA-23, 69	ADC-25	aac(3)-Ia, aadA1, aph(3’’)-Ib, aph(3’)-Ia, aph(3’)-Via, aph(6)-Id		dfrA1, sul1/2, tetB
KAB8	AB (1)	374	NDM-1	ADC-26, OXA-259, CARB-16	aadA1, ant(2’’)-Ia, ant(3’’)-Iia, aph(3’)-Ia	amvA	ble, dfrA1, mphE, msrE, sat2, sul2, tet39
KAB14	AB (1)	164	NDM-1, OXA-58, 91	CARB-16	aac(3)-Iid, ant(2’’)-Ia, aph(3’’)-Ib, aph(3’)-Ia, aph(3’)-Via, aph(6)-Id		floR, mphE, msrE, tet39, sul2
KAB15	AB (1)	164	OXA-420, 91	CARB-16	ant(2’’)-Ia, aph(3’’)-Ib, aph(3’)-Ia, aph(6)-Id		dfrA20, sul2
KEB1	ECL (1)	182	NDM-1	ACT-16, DHA-1 TEM-1B, OXA-1	rmtC, aac(6’)-Ib-cr, aph(3’’)-Ib, aph(3’)-Ia, aph(6)-Id	oqxA/B, mdfA	arr-3, catA2/B3, dfrA14, fosA, mphA, qnrB4, sul1/2, tetA/D
KEB2	ECL (1)	25	NDM-1	DHA-1 ACT-6		oqxA/B, mdfA
KEC1	EC (1)	167	NDM-5	CTX-M-15, OXA-1, TEM-1B	rmtB, aac(6’)-Ib-cr, aadA2	mdfA	catB3, dfrA12, mphA, sul1, tetA
KEC2	EC (1)	167	NDM-5	CTX-M-15, EC, OXA-1	aac(6’)-Ib-cr5, aadA5	acrF, mdtM, emrD	ble, dfrA17, mphA, sul1, tetA
KEC3	EC (1)	648	NDM-5	CTX-M-15, EC, OXA-1, TEM-1	aac(3)-Iia, aac(6’)-Ib-cr5, aac(3)-Iia, aac(6’)-Ib-cr5, aadA5	acrF, mdtM, emrD	ble, catA1, dfrA17, mphA, sul1, tetB
KKPI	KP (1)	147	OXA-181	CTX-M-15, SHV-67, TEM-1B	rmtF, aac(6’)-Ib3, aph(3’’)-Ib, aph(6)-Id	mdfA, oqxA/B	arr-2, dfrA14, fosA, mphA, sul2
KKP2	KP (1)	219	NDM-1	CMY, CTX-M-15, SHV-1	rmtC aac(6’)-Ib3, aadA2, aph(3’)-Ia, aph(3’’)-Ib, aph(6)-Id	oqxA10/B5, kdeA, emrD	ble, dfrA12, floR, fosA, mphA, qnrS1, sul1/2, tetA
KPA2-5	PA (4)	357	NDM-1, OXA-50	OXA-10, PAO,VEB-1	aph(3’)-Iib, aadA1, ant(2’’)-Ia, aac(6’)-Il, aph(3’)-VI		catB7, fosA, sul1, cmlA1, dfrB2, arr-3, tetA
KPA9-13	PA (5)	357	NDM-1	OXA-10, OXA-846, PDC-11, VEB-9	aac(6’)-Il, aadA1, aac(6’)-Il, ant(2’’)-Ia, aph(3’)-Iib, aph(3’)-VI	mexA/E/X	arr-3, ble, catB7, cmlA5, dfrB2, fosA, sul1, tetA
KPA14	PA (1)	357	NDM-1	OXA-396, PDC-3	ant(4’)-Iib, aph(3’’)-Ib, aph(3’)-Iib, aph(6)-Id	mexE	catB7, fosA, sul1
KPA6	PA (1)	654	NDM-1	OXA-396, PAO	aph(3’’)-Ib, aph(6)-Id, ant(4’)-Iib, aph(3’)-Iib		catB7, fosA, sul1
KPA1	PA (1)	316	VIM-1	OXA-10, OXA-395, PAO	aph(3’’)-Ib, aph(6)-Id, aph(3’)-Iib, aac(6’)-Ib3		catB7, fosA, sul1, tetG, floR
KPA7	PA (1)	1203	VIM-6	OXA-10, OXA-395, PAO	aph(3’)-Iib, aadA1, ant(2’’)-Ia, aac(6’)-Ib3		catB7, fosA, sul1, qnrVC1, dfrA5, dfrB5, ereA
KPA8	PA (1)	1203	VIM-6	OXA-10, OXA-395, PAO	aph(3’)-Iib, aadA1, ant(2’’)-Ia, aac(6’)-Ib3		catB7, fosA, sul1, qnrVC1, dfrA5/B5, ereA

PA, P. aeruginosa; KP, K. pneumoniae; EC, E. coli; ECL, E. cloacae; AB, A. baumannii. Antibiotic resistance gene families: AmpC: CMY, ADC-25, ACT-16, DHA-1, ESBL: CTX-M, SHV, TEM. Antibiotic resistance gene targets: Bleomycin-ble; Rifampicin-arr-2, arr-3; Chloramphenicols-catA1, catA2,catB3, catB7, cmlA5, cmlA1, floR; Fosfomycin-fosA;Tetracylines- tetA, B, D, G, and 39; Sulfonamides—sul1and 2; Erythromycin—ereA; Trimethoprim—dfrA1, A5, A12, A14, A17, A20, B2, and B5; Quinolones—qnrVC1, S1, and B4; Macrolides—msrE, mphA, and E; Streptothricin—sat2.

*AmpC genes are highlighted in bold.

^#The16srRNA methyltransferases are highlighted in bold.

The oxacillinases were the most abundant carbapenemases (23/79, 29.1%) detected mostly in A. baumannii isolates. Among the A. baumannii isolates, the predominant genes were bla_{OXA-23, 66, and 69}, with only a few isolates bearing bla_OXA-58 and bla_OXA-91. The bla_OXA-181 and bla_OXA-50 genes were detected in K. pneumonia and P. aeruginosa isolates, respectively. Among the Metallo- β–lactamases, bla_NDM1 (21/79, 26.6%) was detected in A. baumannii, E. cloacae, P. aeruginosa, and K. pneumoniae species. bla_VIM-1 and bla_VIM-6 genes were detected in three P. aeruginosa isolates and bla_NDM-5 genes in three E. coli isolates. The greatest diversity of carbapenemases was found among A. baumannii (bla_NDM-1, bla_{OXA-23, 58, 66, 69, 91},) and P. aeruginosa (bla_{VIM-1, 6,} bla_NDM-1, bla_OXA-50) (Table 3). Although most isolates had a single carbapenemase gene, five A. baumannii isolates (bla_OXA-23/ bla_NDM-1 (KAB13, 25, 26, 27) and bla_NDM1/bla_OXA-58 (KAB 14) and four P. aeruginosa isolates (KPA2-5) (bla_NDM-1, bla_OXA-50) (Table 4) bore both a Class B and D carbapenemase gene. Genes for AmpC were detected among 17/27 A. baumannii (bla_ADC-25), E. cloacae (bla_DHA-1 and bla_ACT-16), and K. pneumoniae (bla_CMY) isolates (Table 4).

Sequence types and geographical distribution of the C-NS isolates

Carbapenemases were identified in all the study counties. However, the geographical distribution of the CR genes, bacterial species, and strain types were variable (Fig 1). Among the C-NS bacterial species, A. baumannii was the most broadly distributed across the counties, while E. cloacae and P. aeruginosa were only detected in Nairobi. Kisii and Nairobi counties had the most types of carbapenemases. The bacterial strain types and their characteristics varied between counties as described for each species in the following section.

Fig 1

The geographical distribution of the carbapenem non-susceptible gram-negative bacteria and carbapenemase genes in five Kenyan counties.

The map of Kenya with the study counties highlighted. The pie charts indicate the distribution of bacterial species, carbapenemase genes identified and the number of each isolate type detected. This custom map was generated in-house using the ArcGIS software Version 10.3.1 (Environmental Systems Research Institute, Red-lands, CA, USA).

Acinetobacter baumannii

The C-NS A. baumannii isolates belonged to four STs: 1, 2, 164, and a novel type. The isolate with a novel ST possessed a new single variant allele of the rplB gene (rplB 213) and was assigned to ST1475. In this study, all twelve ST1 A. baumannii isolates were detected in counties in western Kenya. They are predominantly associated with community-acquired infections (CAI) and were the least drug-resistant of the A. baumannii STs (Fig 1 and Table 1).

The ST1 isolates were mostly susceptible to fluoroquinolones, tetracyclines, tigecycline, and minocycline. A majority of ST1 isolates had the bla_OXA-23 and bla_OXA-69 carbapenemase and the bla_ADC-25 genes. The exception was one isolate (KAB16), which had the bla_NDM-1 carbapenemase, bla_CARB-16, and genes not found in the other ST1 isolates conferring resistance to macrolides, aminoglycosides, and fluoroquinolones. In contrast, the eleven ST2 isolates were multidrug-resistant, predominantly hospital-associated (8/11), and originated from the same hospital in Nairobi (10/11). These ST2 isolates expressed the bla_OXA-23 and bla_OXA-66 carbapenemase, the AmpC gene bla_ADC-25, and the 16s rRNA methyltransferase armA (Table 2), among other resistance genes.

The ST164 A. baumannii isolates (KAB 14, 15) differed in antibiotic resistance profiles and gene composition. The community-acquired KAB14 from Kisii had the bla_NDM1 and bla_OXA-58 carbapenemases and more resistance genes than KAB15 from Kisumu. This healthcare-associated KAB15 had fewer resistance genes and the bla_OXA-420 carbapenemase gene. The ST374 isolate (KAB8) from an HAI in Kisumu was multidrug-resistant and unique because it lacked the oxacillinases. The ST1475 isolate (KAB13) associated with bla_NDM1 and bla_OXA-23 was isolated from an HA-SSTI infection in Kisii. The isolate was susceptible to only the last line drug tigecycline representing a localized high-risk MDR strain.

Enterobacter cloacae

The two E. cloacae isolates, both isolated from Nairobi, belonged to ST182 and ST25. They expressed the bla_NDM1 carbapenemase, bla_DHA-1, bla_ACT-6, mdfA, oqxA, oqxB genes, and were associated with HAIs. ST25 (KEB2) was the least resistant isolate, susceptible to levofloxacin and tigecycline. ST182 (KEB1) was resistant to all drugs tested except tigecycline. It had many drug resistance genes, including the potent 16S methyltransferase rmtC contributing to the highly drug-resistant phenotype.

Escherichia coli

All three E. coli isolates harbored bla_NDM-5. However, the isolates from Nairobi (KEC1, 2) were ST167, while the Kisii isolate (KEC3) was ST648. All the E. coli isolates were only susceptible to tigecycline among the tested antibiotics, although ST648 had more antibiotic-resistant genes than the ST167 isolates implying a greater resistance level.

Klebsiella pneumoniae

ST219 and ST147 K. pneumoniae isolates were identified in Kilifi and Nairobi hospitals (Fig 1), respectively. The ST147 isolate from a CAI expressed a bla_OXA-181 carbapenemase and was resistant to all drugs tested. On the other hand, K. pneumoniae ST219 had the bla_NDM-1, bla_CMY, and the 16S methyltransferase rmtC genes. It was susceptible to levofloxacin, minocycline, and tigecycline.

Pseudomonas aeruginosa

Thirteen of the fourteen P. aeruginosa isolates were resistant to all antibiotics tested. Four ST types, 357, 654, 316, and 1203 –were identified among C-NS P. aeruginosa isolates from Nairobi. All ST357 isolates and the single ST654 isolate (KPA6) had the bla_NDM1 carbapenemase. The single P. aeruginosa ST316 isolate, for which there was limited demographic information available, expressed bla_VIM-2. Of the two isolates identified as ST1203 (KPA 7, 8), both had the bla_VIM-6 gene and genes not identified in any of the other STs of P. aeruginosa in this study: qnrVC1, dfrA5, dfrB5, ereA genes.

Discussion

This study aimed to identify the diversity and distribution of carbapenemase genes among GNB bacteria from an ongoing surveillance study in six hospitals across Kenya. The analysis detected eleven different carbapenemases among five clinically significant bacteria species. Five of the eleven CR genes detected were previously undescribed in Kenya: bla_{OXA-420, 58,181}, bla_NDM-5, and bla_VIM-6. Notably, bla_VIM-6 has not been reported before in Africa since it was first observed in Singapore in 2004 [35] and subsequently spread across Asia [36]. Neither has bla_NDM-5 been reported in East Africa. Other carbapenemase genes identified are naturally occurring or widespread in their species, such as the class D oxacillinases bla_OXA-23 and bla_{OXA-66 and 69} in A. baumannii [37]. One ST374 isolate (KAB8) was unique because it lacked these intrinsic oxacillinases.

Previous studies on carbapenemases in Kenya have focused mostly on Enterobacteriaceae [18]. Our findings show that A. baumannii was the most abundant and widely distributed of the C-NS bacteria carrying approximately 72% of the CR genes detected. It was followed by P. aeruginosa, with approximately 19%. The two species had the most carbapenemase gene diversity. As A. baumannii and P. aeruginosa are significant causes of HAI, their carriage of CR genes could contribute to hard-to-treat hospital outbreaks across broad geographical settings in Kenya—a challenge the World Health Organization (WHO) recognizes [38]. A substantial number of infections were among inpatients with SSTI and catheter-associated UTIs. These indicators stood out as potential risk factors for the acquisition of C-NS infections. Surprisingly, a third of the infections caused by C-NS bacteria were CAI, consistent with the trend witnessed in other parts of the world [39] deviating from the previously strong association of C-NS infections with HAIs. To avoid missing community circulation of C-NS bacteria, CAI should be included in surveillance efforts.

This study also detected various AmpC genes (bla_ADC-25, bla_CMY, bla_DHA-1, and bla_ACT-16), which have not previously been reported to coexist with carbapenemase genes in Kenya, and ESBL genes (bla_CTX-M, _{SHV, and TEM}). Hyper-production of these genes combined with mutations in porin genes or efflux pumps that reduce the influx or increase the efflux can contribute to the carbapenemase resistant phenotype [40]. It is necessary to include all these contributing factors in CR surveillance because, even in the absence of carbapenemases genes, they can cause carbapenem treatment failure.

Multiple antibiotic resistance genes (some of which were not phenotypically tested in this study) and efflux pumps (whose substrates can include antibiotics of multiple classes) coexisted in the C-NS bacteria. These data indicate the breadth of drug resistance and the therapeutic challenge posed by C-NS bacteria. Aminoglycosides are reported to be the third most prescribed drug class after cephalosporins and penicillins in a Kenyan referral hospital [41]. They are often used in combination with other antibiotic classes to treat severe infections. The high rate of aminoglycoside resistance, reflective of this prescription pattern, and the presence of the pan-aminoglycoside resistance genes among all isolates render this drug class ineffective against most C-NS infections. The 16srRNA methyltransferase genes have been previously associated with CR genes, particularly bla_NDM-1 and bla_OXA [42, 43], similar to the findings in this study where 16srRNA methyltransferase was detected alongside bla_OXA-23/66, bla_NDM-1, bla_OXA-181, and bla_NDM-5 genes.

The multidrug-resistant phenotypes in most C-NS isolates suggest an inevitable rise in untreatable GNB infections unless urgent measures are taken to curb these infections. This risk is evident in multiple MDR A. baumannii ST2 isolates identified in one hospital, indicating localized, clonal spread within the hospital. This pattern suggests a prolonged outbreak, as previously reported in a Nairobi Hospital [44]. Carbapenem, considered a last-resort antibiotic, is used to treat MDR infections and is the most commonly prescribed antibiotic in a study in a hospital in Nairobi [45]. As higher-level drugs are often unavailable or too expensive in Kenya, C-NS infections pose a serious challenge. This study shows that the most effective drugs against C-NS infections are tigecycline and minocycline. However, the innate resistance of P. aeruginosa and two A. baumannii isolates resistant to both drugs further limit the therapeutic options.

Few studies in Kenya have identified the strain types of C-NS bacteria. This study has set the baseline for tracking and detecting C-NS STs in Kenya by identifying the C-NS bacteria’s strain types. For example, the identification of A. baumannii STs (1, 2, 164, and a novel ST1475 type) was different from the OXA-23 producing strains identified in a study in a referral hospital in Kenya (ST110, 92, and 109) [44] indicating the broad strain diversity in the C-NS A. baumannii population in Kenya. A. baumannii ST1 and ST2 are global clonal strains associated with multidrug resistance [46]. ST164 is a rare strain type recently reported in Sudan, Brazil, and Turkey [47]. The ST164 A. baumannii bearing bla_OXA-58 and bla_OXA-420 carbapenemases are typically plasmid-mediated and geographically limited to Europe [48], Tunisia [49], and Nepal [50]. These ST164 isolates that carry potentially plasmid-mediated CR genes could be efficient disseminators of CR and MDR among the community and hospital settings in which they were found in Kenya. The A. baumannii ST1475 was previously un-typed, representing a novel strain first identified in Kenya. Carbapenem-resistant P. aeruginosa (CRPA) isolates carrying bla_VIM-2 have been identified in an un-typed Kenya isolate [26] and ST244 and ST640 isolates in Tanzania and throughout Africa, often occurring in outbreaks [51–56]. Other CRPA ST316 isolates have been identified in China expressing the IMP-9 gene [57], but the isolate in this study represents a new case of a bla_VIM-2 ST316 CRPA.

This study observed that different strain types had different drug resistance profiles making it easier to identify the most important strains for close monitoring. Based on the resistance profiles of the CR strains, the study identified high-risk MDR strains of A. baumannii (ST1475, ST2), E. cloacae (ST182), K. pneumoniae (ST147), P. aeruginosa (ST357, 654), and E. coli (ST167, ST648). A. baumannii ST1475 is a recognized international high-risk clone associated with multidrug resistance and hyper-virulence (44). P. aeruginosa ST654 isolates have been identified in Europe [58, 59], Singapore [60], Tunisia [61], and South America [62] associated with KPC, VIM, and IMP carbapenemases. Apart from this study, an extensively drug-resistant ST654 bla_NDM1-producing P. aeruginosa isolate has only been reported in Canada [63]. NDM-5 was first detected in 2011 in an E. coli ST648 strain from India described as a highly virulent and MDR strain [64]. Since then, NDM-5 has been identified globally in different E. coli and K. pneumoniae STs indicating the rapid horizontal transfer of the gene across species. E. coli ST167 is a globally disseminated clone in human and animal populations associated with both multiple resistance and hyper-virulence genes. NDM-5 harboring strains have been described in Europe [65, 66] and China from humans [67]and poultry [68]. The potential zoonotic spread of these bla_NDM-5 E. coli ST167 and ST648 strains in the Kenyan community poses a considerable risk to animal and human populations. The high-risk bla_NDM-1 MDR E. cloacae ST182 isolate has been associated with outbreaks in several countries related to transmissible plasmid-borne bla_NDM-1 [65, 69, 70]. These high-risk strains warrant close monitoring in Kenya as they could act as disseminators of carbapenemases.

An example of the potential or spread of carbapenemase genes is the increasing detection of bla_NDM-1, previously identified in K. pneumoniae, A. baumannii, and P. aeruginosa from Nairobi and Kilifi [22–24, 71, 72]. This distribution is mirrored by this study’s findings of bla_NDM-1 K. pneumoniae and P. aeruginosa in the two counties. However, what was different is that bla_NDM-1 was also detected in E. cloacae isolates (Nairobi) and in A. baumannii (Nairobi, Kisii, and Kericho), an indication of a broader distribution of the CR gene. There have been few reports to date of P. aeruginosa expressing bla_NDM-1 in sub-Saharan Africa and only one in Kenya [71]. It was surprising that five of the eight P. aeruginosa isolates in this study had bla_NDM-1 as P. aeruginosa is most often associated with the Metallo-β lactamases VIM and IMP [73]. But NDM-1 expressing strains, typically associated with ST235, are increasing worldwide since they were first reported in 2011 [74] in Europe [75], India [76], Northern Africa [77] and North America [63] and may also become widespread in Kenya.

The study outcomes suggest that differences in CR’s type and scale among bacterial species exist between hospitals and counties. The greatest number and diversity of C-NS isolates were observed in the two referral hospitals in Kisii and Nairobi. Referral hospitals have factors that contribute to a greater risk of MDR infections, such as more hospital-acquired infections, extended hospitalization stays, invasive devices, and more frequent use of third-and fourth-line drugs [45]. These factors could explain why more carbapenemase genes were detected in Nairobi and Kisii hospitals than in the other lower-level hospitals with less than four carbapenem genes.

The study had some limitations. Only forty eight isolates representing all the available carbapenemase isolates detected over the study period were studied. Additional C-NS GNB isolates will be explored in the future within this ongoing surveillance program to improve the understanding of the geospatial distribution, emergence, spread, and evolution of CR in Kenya. The susceptibility testing was performed on a limited panel of antibiotics on the VITEK2 platform, and the AST results were not confirmed using an independent method. However, the detection of carbapenemase and other antibiotic resistance genes by WGS confirmed the observed resistance phenotypes. The primary concern with CR is the possibility of transmissible versus chromosomal carbapenemases. In this study, detailed analysis to detect the genomic location of the CR genes (chromosomal or plasmid) was not performed as it exceeded its scope, whose main goal was to describe the presence and distribution of carbapenemase genes. These analyses will be the focus of future studies to evaluate the dissemination risks within and between bacterial species.

Conclusions

In conclusion, while CR and the presence of carbapenemase genes have been recognized in Kenya, this study expands our understanding in several ways outlined below.

First, the study has described the diversity of CR genes among clinically significant gram-negative bacteria, including for the first time in E. coli and Enterobacter spp. Second, the research has shown the wide geographical spread of CR across Kenya, including three counties (Kisumu, Kisii, and Kericho) not previously studied. Third, the study provides the first report of carbapenemases OXA-420, 58,181, and VIM-6 and the simultaneous carriage of both carbapenemase genes and AmpC genes in Kenyan isolates. Fourth, CR is identified as a more significant challenge in larger referral hospitals where risk factors such as numbers of inpatient and critical care populations and greater use of third- and fourth-line antibiotics exist. Fifth, the study has made clear that across all hospitals, C-NS A. baumannii warrants attention as its significance in the CR landscape was not previously appreciated. Sixth, the notion that CR is only a nosocomial challenge is dispelled by the C-NS GNB detected in both community and healthcare-associated isolates. Finally, the study has identified STs of globally disseminated high-risk and multidrug-resistant isolates that should be specifically targeted for close monitoring. These isolates are reservoirs and possible transmitters of multiple-drug resistance.

These data highlight the importance of CR surveillance to adequately measure the scale of the problem and identify high-risk strains and emerging resistance genes to track the spread of resistance. Based on this data, measures such as improving infection control and implementing antibiotic stewardship should be implemented with the urgency required to reduce the spread of resistance, limit the morbidity and mortality associated with carbapenem resistance, and to preserve this critical drug class in Kenya.