Drug resistance phenotypes and genotypes in Mexico in representative gram-negative species: Results from the infivar network

Aim This report presents phenotypic and genetic data on the prevalence and characteristics of extended-spectrum β-lactamases (ESBLs) and representative carbapenemases-producing Gram-negative species in Mexico. Material and methods A total of 52 centers participated, 43 hospital-based laboratories and 9 external laboratories. The distribution of antimicrobial resistance data for Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae complex, Acinetobacter baumannii complex, and Pseudomonas aeruginosa in selected clinical specimens from January 1 to March 31, 2020 was analyzed using the WHONET 5.6 platform. The following clinical isolates recovered from selected specimens were included: carbapenem-resistant Enterobacteriaceae, ESBL or carbapenem-resistant E. coli, and K. pneumoniae, carbapenem-resistant A. baumannii complex, and P. aeruginosa. Strains were genotyped to detect ESBL and/or carbapenemase-encoding genes. Results Among blood isolates, A. baumannii complex showed more than 68% resistance for all antibiotics tested, and among Enterobacteria, E. cloacae complex showed higher resistance to carbapenems. A. baumannii complex showed a higher resistance pattern for respiratory specimens, with only amikacin having a resistance lower than 70%. Among K. pneumoniae isolates, blaTEM, blaSHV, and blaCTX were detected in 68.79%, 72.3%, and 91.9% of isolates, respectively. Among E. coli isolates, blaTEM, blaSHV, and blaCTX were detected in 20.8%, 4.53%, and 85.7% isolates, respectively. For both species, the most frequent genotype was blaCTX-M-15. Among Enterobacteriaceae, the most frequently detected carbapenemase-encoding gene was blaNDM-1 (81.5%), followed by blaOXA-232 (14.8%) and blaoxa-181(7.4%), in A. baumannii was blaOXA-24 (76%) and in P. aeruginosa, was blaIMP (25.3%), followed by blaGES and blaVIM (13.1% each). Conclusion Our study reports that NDM-1 is the most frequent carbapenemase-encoding gene in Mexico in Enterobacteriaceae with the circulation of the oxacillinase genes 181 and 232. KPC, in contrast to other countries in Latin America and the USA, is a rare occurrence. Additionally, a high circulation of ESBL blaCTX-M-15 exists in both E. coli and K. pneumoniae.


Introduction
National and local surveillance of drug resistance and the involved genotypes is fundamental to implementing adequate infection control measures [1,2].
The prevalence of carbapenemases from Ambler class A, B, and D, cephalosporinases (AmpCs), is rapidly increasing among Gram-negative bacteria and is rapidly increasing among Gram-negative bacteria and is now widely distributed [3,4].
Some research groups from Mexico have published the drug resistance rates and involved genes for some Gram-negative bacteria, including A. baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, K. pneumoniae, and Escherichia coli [6][7][8][9]. However, information available is limited, and nationwide studies are needed.
To contribute to the study of drug resistance in Mexico, the Network for the Research and Surveillance of Drug Resistance (Red Temática de Investigación y Vigilancia de la Farmacorresistencia INVIFAR, in Spanish) was created in 2018 and has reported an increase in drug resistance for several bacterial species, underlying the increase in carbapenem resistance for Enterobacter spp. and Klebsiella spp. [10,11].
This report presents phenotypic and genetic data on the prevalence and characteristics of ESBL and carbapenemase-producing representative Gram-negative species in Mexico during the first trimester of 2020.

Participating centers, data collection, and analysis
A total of 52 centers participated: 43 hospital-based laboratories and 9 external laboratories.
Identification and susceptibility test results from January 1 to March 31, 2020, from participating laboratories were deposited into the WHONET 5.6 platform and converted to the WHONET using the BacLink 2 tool. WHONET files were analyzed using macros to facilitate the revision, and only one strain per patient was included. The distribution of antimicrobial resistance for E. coli, K. pneumoniae, E. cloacae complex, A. baumannii complex, and P. aeruginosa was analyzed in clinical specimens such as urine, blood, and respiratory specimens. The results were scored according to the Clinical and Laboratory Standards Institute (CLSI) criteria in all laboratories [12].

Included isolates
Participating laboratories sent to the coordinating laboratory all recovered isolates with the following characteristics: carbapenem-resistant Enterobacteriaceae (any species); ESBL or carbapenem-resistant E. coli collected from urine or blood; ESBL or carbapenem-resistant K. pneumoniae recovered from urine, respiratory specimens (endotracheal and bronchoalveolar lavage), or blood; carbapenem-resistant A. baumannii complex and P. aeruginosa recovered from urine, respiratory specimens, or blood. Clinical isolates collected from January 1 to March 31, 2020, were included.
All identifications were confirmed at the coordinating laboratory using MALDI-TOF. After confirmation, phenotypic tests and genotyping tests were performed for each strain.

Beta-lactamase identification and characterization in Enterobacteriaceae
The ESBL phenotypic detection test was performed using the double disk method recommended by the CLSI for E. coli and K. pneumonia [12]. The molecular detection and characterization of ESBLs were performed for bla TEM , bla SHV , and bla CTX-M genes in selected isolates by PCR using previously described and newly designed primers (S1 Table) [13]. A selection of amplified products was sequenced.
Carbapenemase production in Enterobacteriaceae was detected using the CarbaNP test and modified carbapenem inactivation according to the CLSI [12].
All PCR products were sequenced using a Hitachi analyzer (Applied Biosystems, Hitachi High-Technologies Corporation, Tokyo, Japan). DNA sequences were aligned and edited using BioEdit software (Ibis Bioscience, Carlsbad, CA) and matched in a gene bank (www. ncbi.nlm.nih.gov/genbank).

Ethics statement
The local ethics committee of Hospital Civil de Guadalajara "Fray Antonio Alcalde," Jalisco, Mexico) approved this study with reference number 129/17. Informed consent was waived by the ethics committee because no intervention was involved. All participating institutions agreed with the present study.

Participating centers, data, and collected strains
In this study, 52 centers collected strains and sent them to the coordinating laboratory: 43 hospital-based laboratories and 9 external laboratories. The three-month identification and susceptibility data were obtained from 46 centers (37 hospital-based laboratories and 9 external laboratories). The centers were distributed across 19 Mexican states. The characteristics of hospital-based centers are shown in Table 1.
The results of drug susceptibility for 8,245 strains were included for analysis, and 2,243 clinical isolates were collected at the reference laboratory. A selection of 813 isolates (including isolates from each center and state) was included for phenotypic and genotypic analysis.

Drug resistance
Regarding urine isolates, resistance was higher than 55% for all antibiotics in A. baumannii complex. In P. aeruginosa, the lowest percentage of resistance was for piperacillin/tazobactam  Table 2). Also, 44.9% and 39.3% of E. coli and K. pneumoniae, respectively, were reported to be ESBLs producers. Among blood isolates, A. baumannii showed more than 68% resistance for all antibiotics tested, and P. aeruginosa had 37.1% resistance to meropenem. Among Enterobacteriaceae, E. cloacae showed higher resistance to carbapenems (4.4% for meropenem), whereas K. pneumoniae and E. coli had more than 59% resistance for cefepime (Table 3).
Also, 60% and 49.3% of E. coli and K. pneumoniae, respectively, were reported to be ESBLs producers.
A. baumannii showed a higher resistance pattern in respiratory specimens, with only amikacin exhibiting a resistance less than 70%. In general, K. pneumoniae had higher resistance to antibiotics than E. cloacae (Table 4). Also, 47% of K. pneumoniae isolates were reported to be ESBLs producers.

ESBL phenotype and genotype
A total of 1059 E. coli and 370 K. pneumoniae from selected specimens were received. A selection of isolates was evaluated for further analysis (including representative isolates from each center).
Among isolates selected for analysis, 173/215 K. pneumoniae and 419/425 E. coli were confirmed to be ESBLs using the double disk method. All were screened using PCR to detect ESBL-encoding genes bla TEM , bla SHV , and bla CTX .  A selection of ESBL PCR products were sequenced and most of the bla CTX-M encoding genes were detected to be bla CTX -M-15 (32/34, 94.1%), followed by bla CTX-M-55 (17/34, 50.0%). Among the bla SHV gene, bla SHV-5 and bla SHV-11 (with reported ESBL activity), bla SHV-38 (with reported carbapenemase activity), and bla SHV-171 (with no report of ESBL activity) were detected (Table 5).

Discussion
This report presents phenotypic and genetic data on the frequency and characteristics of ESBL and representative carbapenemase-producing Gram-negative species in Mexico using strains collected from 52 centers in 19 Mexican states. OXA-48-like carbapenemases are important causes of carbapenem resistance and are now the most common carbapenemase in some populations [25]. In Enterobacteriaceae, several variants of bla  have been identified, with bla OXA-181 and bla OXA-232 being the two most common [26,27]. Kinetic properties of these two enzymes had been measured, and both appear broadly similar to bla OXA-48 in their activity, with bla OXA-232 demonstrating better hydrolysis of penicillin [28]. In this study, bla OXA-181 and bla OXA-232 were detected in E. coli. At present, bla OXA-232 has been reported in Mexico in two single-center reports: in E. coli, carrying bla OXA-232 plus bla CTXM-15 [8] and in a case-control-control study in which the infection by bla OXA-232 strains was associated with the previous use of β-lactam/β-lactamase antibiotics (OR, 6.2) [29]. The OXA-181 variant has been associated with other carbapenemase genes, including bla NDM-1 and bla VIM-5 [30]. No previous reports of bla OXA-181 circulation in Mexico were identified in the literature.
Enterobacteriaceae-producing OXA-48-like enzymes are rapidly spreading, and thus, laboratory detection should be optimized. This enzyme has low-level hydrolytic activity against carbapenems and, thus, may not be detected [27]. As detected in this study, bla OXA-48-like genes can co-harbor genes encoding ESBL or AmpC enzymes, or both, which confers nonsusceptibility to aztreonam, extended-spectrum cephalosporins, and carbapenem agents and renders these genes a serious menace [31].  [32][33][34][35][36][37]. However, it has remained uncommon in Enterobacteriaceae in America, with some reports in Canada, the United States, and Latin American countries [38][39][40][41]. In this study, the most frequently detected carbapenemase-encoding gene was bla NDM-1 . The NDM carbapenemase was first described in Mexico in 2013 [6], and since then, several reports have been published about it in the county [8,41]. According to our report, NDM is now the most prevalent carbapenemase in Mexico. This study reports by Mexico the first NDM-1-positive Klebsiella variicola isolates considered an emerging pathogen in humans [42].
Within a few years, KPC producers became global as they were reported in America, Europe, and Asia [32,43]. Interestingly, this enzyme has a lower frequency in Mexico when compared to other Latin American countries, as confirmed by our report [43]. KPC and NDM have received special attention due to limited therapeutic options and high mortality associated with infections caused by strains carrying genes that encode these enzymes [44].
Among P. aeruginosa isolates, 44 out of 93 isolates did not contain any of the screened carbapenemase-encoding genes. The most frequent carbapenem resistance mechanism described in P. aeruginosa is the overexpression of efflux pumps and the loss of the Opr porin [52]. Less frequently, genes encoding carbapenemases have been described as an alternative mechanism, with GES variants and IMP, VIM, and NDM reported. In this study, we did not analyze the overexpression of efflux pumps and porins, but bla GES , bla VIM , and bla IMP genes were detected in approximately half of the strains (49/93) ( Table 8). Similar results were reported in Mexico with a prevalence of 36.2% of carbapenemases (IMP, VIM, and GES types) on P. aeruginosa clinical isolates. These genes have been reported to be chromosomally encoded on embedded class 1 integron arrays [53].
Besides carbapenemase-encoding genes, other important mechanisms conferring carbapenem resistance have been observed, including carbapenem hydrolysis by AmpCs in combination with ESBL enzymes, rendering carbapenem resistance to Gram-negative bacteria [54]. In our study, a high frequency of ESBL-producing Enterobacteriaceae was identified, with the AMPc-encoding gene detected in two strains (Enterobacter xiangfangensis (a member of the E. cloacae complex) and E. coli harboring both bla NDM-1 , bla CTXM-15 , and ampC). The presence of AmpC/ESBL and the exact changes of the porins may significantly affect carbapenem resistance. Thus, these mechanisms need to be considered in future research.
The prevalence of bacterial isolates expressing the ESBL phenotype varies across different geographical regions, with rates from 10% to 58% [55]. ESBLs arise primarily due to mutations in the bla TEM , bla SHV , or bla CTX genes, and at present, the CTX-M type is known to be the most frequent non-TEM, non-SHV ESBL [55]. In our study, 72.25% of ESBL-producing K. pneumoniae isolates and 85.7% of E coli isolates harbored bla CTX-M , confirming the spread of this enzyme.
The presence of CTX-M-type enzymes is relevant because they are readily inhibited by all commercially available β-lactamase inhibitors, including avibactam, vaborbactam, and relebactam [56]; a valuable alternative therapy to the recommended ertapenem regimen.
In this study, the non-ESBL TEM-1 was frequently detected, and SHV was detected with no predominance of any subtype. Worldwide, the prevalence of TEM and SHV has diminished, mirroring the worldwide dissemination of isolates producing CTX-M-type -lactamases [57].
Some of the limitations of this study are that not all states in Mexico participated, and the analysis of porins was not included. Furthermore, we only included some bacterial species involved in ESBL production. Our network will continue to actively survey drug resistance and molecular mechanisms involved.
In conclusion, our report identifies NDM as the most frequent carbapenemase-encoding gene in Enterobacteriaceae Mexico with circulation of the oxacillinase genes 181 and 232. KPC, in contrast to other countries in Latin America and the USA, is a rare occurrence. Additionally, a high circulation of ESBL bla CTX-M-15 existed in E. coli and K. pneumoniae.
Supporting information S1