Carbapenem-Resistant Gram-Negative Bacilli Characterization in a Tertiary Care Center from El Bajio, Mexico

Abstract

Carbapenem-resistant Gram-negative bacilli (CR-GNB) are a major public health concern. We aimed to evaluate the prevalence of CR-GNB and the frequency of carbapenemase-encoding genes in a tertiary referral center from El Bajio, Mexico. A cross-sectional study was conducted between January and October 2022; Gram-negative bacilli (GNB) were screened for in vitro resistance to at least one carbapenem. CR-GNB were further analyzed for carbapenemase-production through phenotypical methods and by real-time PCR for the following genes: bla_KPC, bla_GES, bla_NDM, bla_VIM, bla_IMP, and bla_OXA-48. In total, 37 out of 508 GNB were carbapenem-resistant (7.3%, 95% CI 5.2–9.9). Non-fermenters had higher rates of carbapenem resistance than Enterobacterales (32.5% vs. 2.6%; OR 18.3, 95% CI 8.5–39, p < 0.0001), and Enterobacter cloacae showed higher carbapenem resistance than other Enterobacterales (27% vs. 1.4%; OR 25.9, 95% CI 6.9–95, p < 0.0001). Only 15 (40.5%) CR-GNB had a carbapenemase-encoding gene; Enterobacterales were more likely to have a carbapenemase-encoding gene than non-fermenters (63.6% vs. 30.8%, p = 0.08); bla_NDM-1 and bla_NDM-5 were the main genes found in Enterobacterales; and bla_IMP-75 was the most common for Pseudomonas aeruginosa. The mcr-2 gene was harbored in one polymyxin-resistant E. cloacae. In our setting, NDM was the most common carbapenemase; however, less than half of the CR-GNB showed a carbapenemase-encoding gene.

1. Introduction

Antimicrobial resistance (AMR) rates have become a major concern in global health; if the trend continues this way, AMR could become the leading cause of death by 2050 [1]. Carbapenem-resistant Enterobacterales (CRE), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and carbapenem-resistant Acinetobacter baumannii (CRAB) represent high-priority pathogens [2]. The main problem lies in their resistance mechanisms, as they delay appropriate antibiotic therapy, increase hospital length of stay, and worsen patients’ survival [3].

The phenomenon of multidrug-resistant (MDR) bacteria has been specially attributed to the indiscriminate use of antibiotics and the presence of antimicrobial resistance genes. Carbapenemases are described as the most frequent mechanism by which carbapenem resistance has increased [4,5]; nevertheless, it is not the only one. Efflux pumps and porin mutations are other mechanisms described in non-carbapenemase-producing CRE, CRPA and CRAB [5,6].

Carbapenemases can belong to three molecular classes according to Ambler: class A, e.g., Klebsiella pneumoniae carbapenemase (KPC) enzymes or GES-type enzymes; class B metallo-β-lactamases (MBLs) such as New Delhi MBLs (NDM), Verona integron-encoded MBLs (VIM), or imipenemase (IMP); and class D carbapenem-hydrolyzing oxacillinase (OXA), e.g., OXA-48-like enzymes [7].

The rate of carbapenem resistance is considerably higher for non-fermenters (often >60%) than fermenters (often <10%) due to intrinsic factors, including reduced outer membrane permeability and efficient efflux pumps, even in wild-type strains [8]; however, prevalence of carbapenemase-encoding genes varies widely among studies, ranging from 4 to 32% for CRPA, with predominance of bla_VIM, bla_IMP, and bla_GES, between 45 and 90% for CRE, with clear dominance of bla_KPC and bla_NDM, and up to 80 to 100% for CRAB, the main genes found being bla_OXA-like [9]. Global distribution of carbapenemases has shown a predominance of bla_KPC in North America and Europe; bla_NDM and bla_IMP in some regions of Asia; bla_VIM in Europe; and bla_OXA-48 in the Middle East and Africa [5]. In Mexico, epidemiological data have been expanding in recent years, showing bla_NDM as the most frequent gene in both K. pneumoniae and E. coli, followed by bla_KPC and bla_VIM; among P. aeruginosa, bla_VIM and bla_IMP are the main encoded genes, whereas A. baumannii has a predominance of bla_OXA-24, bla_OXA-40, and bla_OXA-23 [10,11].

As information on carbapenem resistance varies by geographical situation and is constantly evolving, regional surveillance is fundamental for the implementation of adequate infection control measures and provide good antibiotic use policies. We aimed to estimate the prevalence of carbapenem-resistant Gram-negative bacilli (CR-GNB) and characterize the presence of carbapenemase-encoding genes in a tertiary regional referral center from El Bajío, Mexico.

2. Results

2.1. Gram-Negative Bacilli Isolates

A total of 508 cultures with positive growth of Gram-negative bacilli (GNB) isolates (including Enterobacterales and non-fermenters) were included; 360 isolates were from inpatient culturing (70.9%). The isolates were recovered from different clinical specimens: urine in 50.8%, blood 15.9%, respiratory tract 15.9%, wound 14.2%, peritoneal fluid 2%, pleural effusion 1%, and 0.2% from cerebrospinal fluid. The strains isolated corresponded to Escherichia coli in 53.5%, Klebsiella pneumoniae 16.7%, Pseudomonas aeruginosa 14.6%, Enterobacter cloacae 3.5%, Proteus mirabilis 2.4%, Serratia marcescens 1.8%, Morganella morganii 1.6%, Acinetobacter baumannii 1.2%, and Citrobacter freundii 1%, whereas the remaining 3.7% was represented by four Klebsiella oxytoca, four Achromobacter xylosoxidans, three Providencia rettgeri, two Proteus vulgaris, two Pantoea agglomerans; Raoultella planticola, Ochrobactrum anthropi, Klebsiella aerogenes, and Citrobacter koseri contributed with one isolate each. The presence of extended spectrum ß-lactamases (ESBLs) was estimated to be 60.1% (CI 95%, 54.8–65.2); it was identified in 59.9% of E. coli and 60.6% of Klebsiella spp.

2.2. Carbapenem-Resistant Gram-Negative Bacilli

In total, 37 of 508 strains were identified as carbapenem-resistant isolates, and their frequency was estimated to be 7.3% (95% Confidence Interval [CI] 5.2–9.9); 11/428 were CRE (2.6%, 95% CI 1.3–4.5), and 26/80 were carbapenem-resistant non-fermenters (32.5%, 95% CI 22.4–43.9). Twenty-seven were from inpatient samples (72.9%). Carbapenem-resistant isolates were mainly recovered from urine 29.7%, lower respiratory tract (tracheal or bronchial aspirate) 21.6%, wound 21.6%, blood 16.2%, sputum 5.4%, and peritoneal fluid 5.4%. Carbapenem-resistant isolates corresponded to P. aeruginosa in 67.6%, E. cloacae 13.5%, E. coli 10.8%, K. pneumoniae 5.4%, and A. baumannii 2.7% (

Table 1. Origin and bacterial differences between carbapenem-resistant and susceptible isolates.

	Carbapenem-Susceptible Isolates (n = 471)	Carbapenem-Resistant Isolates (n = 37)	p-Value
Age, years (IQR)	48 (27–61)	43 (27–55)	0.405 *
Sex, n (%)			0.004 **
Male	214 (45.4)	26 (70.3)
Female	257 (54.6)	11 (29.7)
Setting, n (%)			0.770 **
Inpatient	333 (70.7)	27 (73)
Outpatient	138 (29.3)	10 (27)
Culture site, n (%)
Urine	247 (52.4)	11 (29.7)	0.007 **
Blood	75 (15.9)	6 (16.2)	1 **
Sputum	24 (5.1)	2 (5.4)	1 ***
Tracheal/bronchial	47 (10)	8 (21.6)	0.047 ***
Wound	64 (13.6)	8 (21.6)	0.177 **
Peritoneal fluid	8 (1.7)	2 (5.4)	0.160 ***
Pleural fluid	5 (1.1)	0	1 ***
CSF	1 (0.2)	0	1 ***
Enterobacterales, n = 428 (%)	417 (88.5)	11 (29.7)
E. coli, n = 272	268 (56.9)	4 (10.8)	0.107
K. pneumoniae, n = 85	83 (17.6)	2 (5.4)	1
E. cloacae, n = 18	13 (2.8)	5 (13.5)	<0.0001
Others, n = 53	53 (11.2)	0	0.373
Non-fermenters, n = 80 (%)	54 (11.5)	26 (70.3)	0.657
P. aeruginosa, n = 74	49 (10.4)	25 (67.6)
A. baumannii, n = 6	5 (1)	1 (2.7)

Comparison between carbapenem-susceptible and carbapenem-resistant isolates. CSF = Cerebrospinal fluid; IQR = Interquartile range. * Mann–Whitney U test. ** Chi-square test. *** Fisher-exact test.

).

Carbapenem Resistance by Culture Site

According to culture site, carbapenem resistance was higher in tracheal/bronchioalveolar samples (14.5% vs. 6.8%; OR 2.5 95% CI 1.07–5.75, p = 0.047) and lower in urine (4.4% vs. 11.6%; OR 0.38 95% CI 0.18–0.79, p = 0.009), compared to all other sites. Non-fermenters had higher rates of carbapenem resistance than Enterobacterales (32.5% vs. 2.6%; OR 18.3, 95% CI 8.5–39, p < 0.0001). Regarding bacterial species, E. cloacae showed higher carbapenem resistance than other Enterobacterales (27% vs. 1.4%; OR 25.9, 95% CI 6.9–95.8, p < 0.0001); among non-fermenters, there was no statistical difference between P. aeruginosa and A. baumannii (33.8% vs. 16.7%, p = 0.657). E. coli showed a higher carbapenem resistance rate in blood cultures than other sites (7% vs. 0.4%, OR 17.1 95% CI 1.7–168, p = 0.01). All other bacteria did not show significant differences by site of culture. Frequency of CR-GNB is shown in

Table 2. Frequency of carbapenemase resistance in Gram-negative according to culture site.

Bacteria	Culture Site						Overall Estimation of Carbapenem-Resistance Prevalence
	Urine	Blood	Sputum	Tracheal/Bronchial	Wound	Peritoneal Fluid
Enterobacterales
E. coli	0/182	3/43	0/6	0/7	1/28	0/3	4/272
	(0%)	(7%)	(0%)	(0%)	(3.6%)	(0%)	(1.5%)
K. pneumoniae	1/30	0/13	0/6	0/18	0/12	1/4	2/85
	(3.3%)	(0%)	(0%)	(0%)	(0%)	(25%)	(2.4%)
E. cloacae	1/3	1/2	0/1	1/6	2/6	0	5/18
	(33.3%)	(50%)	(0%)	(16.7%)	(33.3%)		(27.8%)
Others	0/23	0/9	0/2	0/6	0/11	0/2	0/53
Estimated prevalence for Enterobacterales	2/238	4/67	0/15	1/37	3/57	1/9	11/428
	(0.8%)	(6.0%)	(0%)	(2.7%)	(5.2%)	(11.1%)	(2.6%)
Non-fermenters
P. aeruginosa	9/19	2/12	2/11	6/15	5/15	1/1	25/74
	(47.4%)	(16.7%)	(18.2%)	(40%)	(33.3%)	(100%)	(33.8%)
A. baumannii	0/1	0/2	0	1/3	0	0	1/6
	(0%)	(0%)		(33.3%)			(16.7%)
Estimated prevalence for non-fermenters	9/20	2/14	2/11	7/18	5/15	1/1	26/80
	(45%)	(14.2%)	(18.2%)	(38.9%)	(33.3%)	(100%)	(32.5%)
All
Overall estimation prevalence according to site	11/258	6/81	2/26	8/55	8/72	2/10	37/508
	(4.3%)	(7.4%)	(7.7%)	(14.5%)	(11.1%)	(20%)	(7.3%)

Prevalence of carbapenem-resistant bacilli by culture site and species.

.

2.3. Carbapenemase Production

First, carbapenemase-producing strains were phenotypically identified by the modified carbapenem inactivation method (mCIM) in 15 of the 37 CR-GNB samples (40.5%); of the latter, twelve were inhibited by the ethylenediaminetetraacetic acid (EDTA) carbapenem inactivation method (eCIM), suggesting Ambler class B carbapenemases, and the remaining three samples were negative to eCIM. Secondly, the NG-test CARBA-5^® identified the involved protein in 12 of the 37 CR-GNB samples (32.4%). Lately, carbapenemase-encoding genes were identified by PCR; fifteen isolates (40.5%) were positive for one carbapenemase-encoding gene; and, out of them, seven strains (46.7%) had the bla_NDM gene, four (26.7%) the bla_IMP gene, three (20%) the bla_GES gene, and one (6.6%) the bla_VIM gene. No bla_OXA-like or bla_KPC genes were detected. There were no co-producer genes. Enterobacterales showed a higher rate of carbapenemase-encoding genes than non-fermenters (63.6% vs. 30.8%); however, this result did not reach statistical significance (p = 0.08).

All results were concordant between NG-test CARBA-5^® and PCR, except for bla_GES, which is not included in the NG-test CARBA-5^®. After the DNA sequencing, out of the bla_NDM genes, bla_NDM-1 and bla_NDM-5 were the most prevalent, with 42.9% each, followed by bla_NDM-7 in 14.2%. Among bla_IMP genes, there were three bla_IMP-75 (75%) and one bla_IMP-83 (25%); furthermore, there were two bla_GES-40, one bla_GES-26, and, finally, one bla_VIM-2. The only isolate of E. cloacae resistant to colistin was positive for the mcr-2 gene (

Table 3. Carbapenemase-encoding genes by bacterial species.

Bacterial Specie	Isolates (%)	Carbapenemase Genes				Carbapenemase Producing Isolates (%)
		Class A	Class B
		GES	NDM	IMP	VIM
Enterobacterales
E. cloacae	5 (13.5)	-	2 NDM-1	-	-	2 (40)
E. coli	4 (9.5)	-	3 NDM-5 1 NDM-1	-	-	4 (100)
K. pneumoniae	2 (5.4)	-	1 NDM-7	-	-	1 (50)
Total of CRE	11 (29.7)	-	7 (100)	-	-	7 (63.6)
Non-fermenters
P. aeruginosa	25 (67.6)	2 GES-40 1 GES-26	-	3 IMP-75 1 IMP-83	1 VIM-2	8 (32)
A. baumannii	1 (2.7)	-	-	-	-	0
Total of CRPA and CRAB	26 (70.3)	3 (37.5)	-	4 (50)	1 (12.5)	8 (30.8)
All
Total (%)	37 (100)	3 (8.1)	7 (18.9)	4 (11)	1 (2.7)

Genotype detection of carbapenemase-encoding genes in Gram-negative bacilli. CRE = carbapenem-resistant Enterobacterales, CRPA = carbapenem-resistant Pseudomonas aeruginosa, CRAB = carbapenem-resistant Acinetobacter baumannii.

).

2.4. Antimicrobial Resistance Rates

Resistance to ertapenem was 100%, meropenem 89%, doripenem 89%, and imipenem 84%. Only one E. cloacae showed resistance to colistin. Among these strains, the resistance to at least one aminoglycoside was 57%, 62% for monobactams, 68% for fourth-generation cephalosporins, 49% for fluoroquinolones, and 62% for piperacillin–tazobactam (

Table 4. Antimicrobial resistance of the carbapenem-resistant isolates.

P. aeruginosa (n = 25)
	AMK I = 32 R ≥ 64	GEN I = 8 R ≥ 16	ATM I = 16 R ≥ 32	CAZ I = 16 R ≥ 32		CEF I = 16 R ≥ 32	CIP I = 1 R ≥ 2	LVX I = 2 R ≥ 4	DOR I = 4 R ≥ 8	IMP I = 4 R ≥ 8	MEM I = 4 R ≥ 8	COL I ≤ 2 R ≥ 4	PTZ I = 32/4–64/4 R ≥ 128/4
I	16	12	8	4		8	8	4	0	0	4	100	36
R	24	60	52	52		44	44	40	96	88	92	0	36
E. cloacae (n = 5)
	AMK I = 32 R ≥ 64	GEN I = 8 R ≥ 16	ATM I = 8 R ≥ 16	CAZ I = 8 R ≥ 16	CEF SDD = 4–8 R ≥ 16	CIP I = 0.5 R ≥ 1	LVX I = 1 R ≥ 2	ETP I = 1 R ≥ 2	DOR I = 2 R ≥ 4	IMP I = 2 R ≥ 4	MEM I = 2 R ≥ 4	COL I ≤ 2 R ≥ 4	PTZ SDD = 16/4 R ≥ 128/4
I	0	0	0	0	0	20	0	0	0	0	0	80	0
R	20	40	80	100	40	20	20	100	60	60	60	20	100
E. coli   (n = 4)
	AMK I = 32 R ≥ 64	GEN I = 8 R ≥ 16	ATM I = 8 R ≥ 16	CAZ I = 8 R ≥ 16	CEF SDD = 4–8 R ≥ 16	CIP I = 0.5 R ≥ 1	LVX I = 1 R ≥ 2	ETP I = 1 R ≥ 2	DOR I = 2 R ≥ 4	IMP I = 2 R ≥ 4	MEM I = 2 R ≥ 4	COL I ≤ 2 R ≥ 4	PTZ SDD = 16/4 R ≥ 128/4
I	0	0	25	0	0	0	0	0	0	0	0	100	0
R	25	25	75	100	75	75	75	100	100	100	100	0	100
K. pneumoniae   (n = 2)
	AMK I = 32 R ≥ 64	GEN I = 8 R ≥ 16	ATM I = 8 R ≥ 16	CAZ I = 8 R ≥ 16	CEF SDD = 4–8 R ≥ 16	CIP I = 0.5 R ≥ 1	LVX I = 1 R ≥ 2	ETP I = 1 R ≥ 2	DOR I = 2 R ≥ 4	IMP I = 2 R ≥ 4	MEM I = 2 R ≥ 4	COL I ≤ 2 R ≥ 4	PTZ SDD = 16/4 R ≥ 128/4
I	0	0	0	0	0	0	50	0	0	0	0	100	0
R	50	50	50	100	50	100	50	100	50	50	50	0	100
A. baumannii   (n = 1) *
	AMK I = 32 R ≥ 64	GEN I = 8 R ≥ 16	CAZ I = 16 R ≥ 32	CEF I = 16 R ≥ 32		CIP I = 2 R ≥ 4	LVX I = 4 R ≥ 8	DOR I = 4 R ≥ 8		IMP I = 4 R ≥ 8	MEM I = 4 R ≥ 8	COL I ≤ 2 R ≥ 4	PTZ I = 32/4–64/4 R ≥ 128/4
I	0	0	0	0		0	0	0		0	0	100	0
R	0	100	100	100		100	100	100		100	100	0	100

Results are expressed in resistance percentage (%) according to CLSI 2022. All minimal inhibitory concentration breakpoints are expressed in μg/mL. I: Intermediate; R: Resistant; SDD: susceptible-dose-dependent. Antibiotics tested: AMK: amikacin; ATM: aztreonam; CAZ: ceftazidime; CEF: cefepime; CIP: ciprofloxacin; COL: colistin; DOR: doripenem; ETP: ertapenem; GEN: gentamicin; IMP: imipenem; LVX: levofloxacin; MEM: meropenem; PTZ: piperacillin/tazobactam. * Only 1 isolate of A. baumannii was included.

).

3. Discussion

Globally, many regions have not implemented active surveillance programs to monitor CR-GNB. Our work is the first to characterize carbapenemase genes in the Bajio region of Mexico, expanding the knowledge of the mechanisms of resistance in our country. We found an overall carbapenem-resistance rate in Gram-negative bacteria of 7.3% (95% CI, 5.2–9.9), being 2.6% (95% CI 1.4–3.5) for Enterobacterales and 32.5% (95% CI 22.4–43.9) for non-fermenters, which is comparable with data from Mexico but lower than other international reports [4,9,12].

The University Plan for the Control of Antimicrobial Resistance (PUCRA, Plan Universitario de Control de la Resistencia Antimicrobiana), a national surveillance program, showed a carbapenem resistance rate of 0.4–1% for E. coli, 1–5% for K. pneumoniae, 2–4% for Enterobacter sp., 59% for Acinetobacter sp., and 25% for P. aeruginosa [13]. On a subsequent study by the INVIFAR network in Mexico [11], they reported a low carbapenem resistance in E. coli (<1%) and K. pneumoniae (2.9%) but high carbapenem resistance in E. cloacae (10.9%), P. aeruginosa (38–44%), and A. baumannii (83%). Our center showed similar rates for E. coli (1.5%), K. pneumoniae (2.4%), and P. aeruginosa (33.8%) but a higher prevalence of carbapenem resistance for E. cloacae (33%); although it was lower for A. baumannii (16.7%), it should be noted that the latter was underrepresented in our study. In addition, the INVIFAR network reported that 45% of E. coli and 39% of K. pneumoniae were ESBLs producers, which were much lower compared with our rates of approximately 60%.

The Antimicrobial Surveillance Program SENTRY in Latin America showed that 4.3% of Enterobacterales strains were CRE, with the highest rates in Brazil (9%) and Argentina (6.3%) and the lowest in Chile (0.4%) and Mexico (0.7%) [14]. However, these numbers have evolved in recent years, and our study showed a CRE rate of 2.6%. The GLASS report in 2021 mentioned a worldwide prevalence of CRE up to 10–15% [15]. However, it should be noted that Mexico was not part of the antimicrobial resistance surveillance programs included in this study. On the other hand, the prevalence of carbapenem resistance among non-fermenters in Latin America has been described as high as 66% for P. aeruginosa and up to 90% for A. baumannii [16], discordant to the findings of our study.

Carbapenem resistance is directly influenced by the culture site, being higher in respiratory infections than in bloodstream infections [17]. This was concordant with our analysis, where the highest proportion of CR-GNB were isolated from lower respiratory tract samples, whereas the lowest proportion was for urine cultures.

Given the high lethality rate associated with CRE, CRPA, and CRAB, assessment of the hospital- or ward-level risk of CR-GNB is crucial, especially by defining the specific resistance mechanisms [3]. However, few laboratories can perform routine phenotypic or genotypic testing for carbapenemases; thus, determining the local epidemiology allows for a better choice of treatment [18]. The latter becomes relevant due to the possible use of the new, although not widely available, β-lactam/β-lactamase inhibitors, such as ceftazidime/avibactam, meropenem/vaborbactam, and imipenem/relebactam, against KPC-producing strains [3,18,19]. Nevertheless, the emergence of resistance to ceftazidime/avibactam has been recently described in KPC-3-producing strains [20]; ceftazidime/avibactam or cefiderocol as monotherapy are options against OXA-producing strains [18,21,22]. For MBL-producing strains, there are more limited options, especially the combination of ceftazidime/avibactam with aztreonam or cefiderocol as monotherapy, whereas tigecycline, eravacycline, and polymyxins remain as alternative but more toxic options, regardless of the presence of carbapenemases [18,23]. This becomes a challenge for Mexico since it is a country with a predominance of MBLs [10,11].

The most frequent types of carbapenemase-encoding genes in our study were bla_NDM in Enterobacterales and bla_IMP in P. aeruginosa; however, no bla_OXA or bla_KPC were identified. Worldwide, bla_NDM and bla_KPC are the main carbapenemases found in Enterobacterales;, whereas bla_OXA, bla_IMP, bla_VIM, and bla_GES are primarily found in P. aeruginosa and A. baumannii [5,24,25,26]. Few studies in Mexico have evaluated the presence of carbapenemase-encoding genes in CRE, CRPA, and CRAB. For CRPA, bla_IMP and bla_VIM, followed by bla_GES, are the most commonly described [10,11,27,28], in line with our results. Meanwhile, for CRE, some Mexican authors have described bla_NDM-1 as the most associated gene; however, bla_OXA-48, bla_OXA-181, bla_OXA-232, and bla_KPC-2 have also been reported [10,11,29,30,31]. In addition to bla_NDM-1, we identified isolates carrying the bla_NDM-5 and bla_NDM-7 genes. For CRAB, the most frequent encoding genes described were bla_OXA-24 and bla_OXA-40, followed by bla_OXA-23, bla_VIM, and bla_NDM [11]. It is noteworthy that we had a high proportion of isolates with no carbapenemase-encoding genes (60%) in contrast to previously reported studies [4,12,25,32], suggesting the presence of non-enzymatic mechanisms or carbapenemases other than the ones evaluated, closer to those described by Garza-Gonzalez et al. [11], where 47% was negative for the presences of the carbapenemases tested.

The use of point-of-care tests, such as NG-test CARBA-5^®, is a good complementary option to guide antimicrobial therapy since they have shown excellent performances compared with molecular techniques and provide results within 15–30 min. However, it should be considered that they do not detect all types of carbapenemases, as seen with bla_GES in our sample, and may not perform as well for P. aeruginosa [33].

In addition to allowing better guidance of antibiotic therapy, distinguishment between carbapenemase-mediated and non-enzymatic mechanisms is important since the former has been associated with higher odds of dying at 14 days and can spread more efficiently between patients, requiring an efficient equipment disinfection policy, combined with timely isolation of the cohort to reduce the spread of these strains [9,34]. However, as non-carbapenemase-producing strains not only target carbapenems and β-lactam antibiotics, they usually are associated with MDR. Mutations in AmpC and its regulatory genes are commonly attributed to P. aeruginosa resistance to new β-lactam-β-lactamase inhibitor combinations [35], whereas plasmid-encoded AmpC is most commonly found in resistant Enterobacterales [36]. Among efflux-pump systems, AcrAB-TolC RND is the most commonly described [37]. Finally, porin mutations (especially in OmpC and OmpF) block the entry of carbapenems into the cell [38].

Polymyxin susceptibility remains in almost all cases, except for one E. cloacae with the presence of mcr-2 gene. Despite their high toxicities, polymyxins continue as a last-line pharmacological group in cases of Gram-negative MDR infections. The mcr genes are of great concern due to their high potential for horizontal propagation. Among the variants described, mcr-1 is the most prevalent within Mexico [10] and globally [39]; thus far, mcr-2 has been described in human specimens, especially in Asia [40,41]. However, to the best of our knowledge, this is the first report of mcr-2 in a Mexican patient.

4. Materials and Methods

4.1. Design

A cross-sectional study was carried out between 1 January and 31 October 2022 at the Hospital Regional de Alta Especialidad del Bajío, a 184-bed tertiary regional referral center located in León, Guanajuato, Mexico.

4.2. Sampling

Isolates from clinically relevant sites samples, such as blood, urine, respiratory, cerebrospinal fluid, peritoneal, pleural, and deep-tissue cultures with Gram-negative bacterial growth, were included. All data were collected in the microbiology laboratory at the Hospital Regional de Alta Especialidad del Bajío. Identification was performed with VITEK-2 (Biomérieux, Marcy l’ Etoile, France), and susceptibility tests were performed by microdilution broth method following the Clinical and Laboratory Standards Institute (CLSI) 2018 M07 recommendations [42] and breakpoints according to M100 Ed. 32 (2022) criteria [43]. Duplicated isolates, i.e., more than one isolate per patient in the same hospitalization, were identified and discharged of the database. Bacteria with resistance to at least one carbapenem (meropenem, imipenem, doripenem, and ertapenem; this last one was not considered for Pseudomonas aeruginosa) were selected for analysis. Bacteria from single colonies were stored at −20 °C in brain heart infusion broth with 15% of glycerol until used. An internal protocol (HRAEB-POE-013) for type B biological substances (UN 3373) was used to send the samples to the infectious disease laboratory at the Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra in Mexico City.

4.3. Tests

We performed two phenotypic tests to screen for carbapenemase-producing isolates. First, we performed mCIM (modified carbapenemase inactivation method). To identify metallo ß-lactamase-producing strains, we used the mCIM variant, with EDTA (ethylenediaminetetraacetic acid) as a chelator (eCIM) [42]. Once the producer strain was identified, the next step was to identify the protein involved through NG-test CARBA-5^® [33] (KPC, OXA-48, VIM, IMP, and NDM). (eCIM). All these isolates were further analyzed by a polymerase chain reaction (PCR) to identify the following carbapenemase-encoding genes: bla_KPC, bla_GES, bla_NDM, bla_VIM, bla_IMP, and bla_OXA-48. An Enterobacter cloacae was shown to be resistant to colistin; therefore, it was decided to be screened for mobile colistin resistance (mcr) genes. The gene sequences used are listed in

Table 5. Gene sequences used for polymerase chain reaction.

Gen	Sequence	Amplicon (bp)
blaGES	Forward 5′-TCATTCACGCHCTATTVCTGGCA-3′ Reverse 5′-CTATTTGTCCGTGCTCAGG-3′	857
blaKPC	Forward 5′-ATGTCACTGTATCGCCGTCT-3′ Reverse 5′-TTACTGCCCGTTGACGC-3′	798
blaNDM	Forward 5′-ATGGAATTGCCCAATATT-3′ Reverse 5′-TCAGYGCAGCTTGTCGGC-3′	650
blaVIM	Forward 5′-AGATTGVCGATGGTGTTTGGT-3′ Reverse 5′-GAGCAAGTCTAGACCGCCC-3′	430
blaIMP	Forward 5′-GTTTATGTTCATACTTCGTTTG-3′ Reverse 5′-CAACCAGTTTTGCHTTAC-3′	425
blaOXA-48	Forward 5′-GAATGCCTGCGGTAGCAA-3′ Reverse 5′-AAACCATCCGATGTGGGCAT-3′	438
mcr-1	Forward 5′-TCTTGTGGCGAGTGTTGCCGT-3’ Reverse 5′-CCAATGATACGCATGATAAACGCTG-3′	190
mcr-2	Forward 5′-CTGTTGCTTGTGCCGATTGGACTA-3′ Reverse 5′-ACGGCCATAGCCATTGAACTGC-3′	210

.

4.3.1. Carbapenemase Related-Genes Detection

Bacterial DNA was extracted by thermal shock (95 °C/20 min) using PBS 1×. Next, the tubes were centrifuged (10,000 rpm/20 min), and the supernatants were separated in new tubes. PCR conditions were the following: 10× PCR Buffer (New England BioLabs, Ipswich, MA, USA) containing 2 mM of MgCl₂, 0.2 mM of each of the dNTPs (Invitrogen, Waltham, MA, USA), 10 pmol of each oligonucleotide (Table 5), 1.5 U of Taq polymerase (BioLabs, USA), and 3 uL of DNA.

The amplification was carried out using the following program: 1 cycle of 5 min at 95 °C, 35 cycles of 50 s at 95 °C, 60 s at 55 °C, and 50 s at 68 °C, followed by 1 final cycle of 10 min at 68 °C (Veriti, Applied Biosystems, Waltham, MA, USA); Tm for bla_IMP was 52 °C, Tm bla_oxa-48, bla_VIM, bla_GES, bla_NDM, and bla_KPC was 55 °C; Tm for mcr-1 and mcr-2 was 54 °C.

PCR products were separated in a 1% agarose gel at 120 volts for 40 min, and SYBRGreen (Invitrogen, USA) was used such as DNA intercalant. Bands were visualized in Gel Doc XR+ (BioRad, Hercules, CA, USA) with Image Lab Software version 6.1 (BioRad, Hercules, CA, USA).

4.3.2. Sequencing

Prior to the sequencing process, re-amplification and labeling using the BigDye^® Direct Cycle Sequencing Kit (Thermo Scientific, Waltham, MA, USA) were performed. Sequencing was performed using the two oligonucleotides (described for the amplification). A 3730xl DNA Analyzer (Applied Biosystems, USA) was used for sequencing, using POP-7 (Thermo Scientific, USA). GenBank was used to compare the sequences obtained with those of reference organisms in this database [44]. Identification was corroborated by performing independent sequencing for each primer.

4.4. Statistical Analysis

SPSS software version 25.0 was used for statistical analysis. A descriptive analysis of the distribution of all Gram-negative strains at the hospital was performed, as well as an analysis of the subpopulation of carbapenem-resistant Gram-negative strains. Groups were established according to the site where the sample was obtained and the microorganism isolated. The absolute and relative frequencies for each group and their relationships with the antibiotic resistance profile and the presence of carbapenemases were described. A comparative analysis between groups was performed. p-values were obtained using the chi-square test (x²) or Fisher’s exact test for qualitative variables, according to their expected values. For quantitative variables, the Student’s t test or Mann–Whitney U test was used according to their distribution, which was evaluated by the Shapiro–Wilk test. The confidence interval will be 95%, with a p-value required for statistical significance <0.05.

4.5. Limitations

Despite being a regional referral center, our results may not be applicable in primary care settings, as our patients were more likely to have previous hospitalizations and antibiotic exposures due to their underlying conditions. We did not evaluate all the described carbapenemase-encoding genes. Also, porin and efflux-pump mutations were not assessed; therefore, we could not attribute with certainty the mechanism of resistance in some strains. As we did not evaluate our population throughout time, future research should focus in this direction to analyze the CR-GNB trends in our country.

5. Conclusions

In the region of El Bajio, Mexico, the prevalences of carbapenem-resistant strains are 2.6% for Enterobacterales and 32.5% for non-fermenters. Less than half of CR-GNB showed a carbapenemase-encoding gene. bla_NDM was the most frequent carbapenemase-encoding gene for Enterobacterales, whereas bla_IMP-75 was for P. aeruginosa. Continuous surveillance is necessary to provide adequate control measures and improve antibiotic stewardship. E. cloacae in our setting will be of special attention since we reported the first Mexican isolate harboring the mcr-2 gene.