Detection of carbapenemase-producing bacteria in a public healthcare center from Venezuela

Introduction: The dramatic increase in the prevalence and clinical impact of infections caused by Carbapenemase-Producing Bacteria in the nosocomial setting in Latin America represents an emerging challenge to public health. The present study detected carbapenemase-producing Gram-negative bacteria in patients from a Hospital from Venezuela, by phenotypic and genotypic methods. Methodology: The bacterial identification was carried out using conventional methods. The resistance to carbapenems was performed by KirbyBaüer disk diffusion method, according to CLSI recommendations. The modified Hodge Test, double-disk with phenylboronic acid, doubledisk with EDTA and Blue Carba Test were performed to detect phenotypic carbapenemase producers. The carbapenemase-encoding genes blaKPC, blaVIM, blaIMP, blaOXA-2, blaOXA-3, blaOXA-15 and blaOXA-21 were determined. Results: The bacterial species identified were Klebsiella pneumoniae complex (181), Pseudomonas aeruginosa (51), and Acinetobacter baumannii-calcoaceticus complex (119). KPC-type was detected in 40.17% of isolates and VIM-type in 14.53%. KPC-type gene was only identified in K. pneumoniae isolates (77.9%). VIM-type gene was identified in P. aeruginosa (86.27%) and K. pneumoniae isolates (3.87%). There was not detection of IMP-type and OXA-type genes. Conclusions: We found a predominance of K. pneumoniae KPC producers and a high rate of VIM-producing P. aeruginosa. The epidemiology of CPB in Venezuela is rapidly evolving, and enhanced surveillance and reporting are needed across the healthcare continuum.


Introduction
The spread of Carbapenemase-Producing Bacteria (CPBs), a major public health threat, continues to increase on a global level and is associated with significant morbidity and mortality [1,2]. Carbapenemases belonging to all of the three classic classes (A, B and D) have been identified all over the Latin American region [3].
The Clinical Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines are the most popular breakpoint guidelines used in antimicrobial susceptibility testing worldwide. Several studies show acceptable level of agreement between EUCAST and CLSI zone diameter breakpoints to carbapenems for Gram-negative bacilli and laboratories with similar antibiotic susceptibility patterns may choose to adopt either guideline without fear of significantly altering reported antibiotic susceptibility [11,12].
The increased frequency of reports on carbapenemases in Latin America and the Caribbean suggests they have successfully spread and become endemic in some countries [13]. The detection of carbapenemase-mediated carbapenem resistance is essential for patient management, infection control, and public health efforts [14]. Currently, characterization of the underlying mechanism of carbapenem resistance is not undertaken by most clinical microbiology laboratories for therapeutic decision-making. However, understanding if an organism is carbapenemase producing and, if so, the class of carbapenemase (s) produced has treatment implications [15]. Therefore, the aim of this study was to detect KPC, VIM and OXA carbapenemases in carbapenem-resistant Gramnegative bacteria in a Hospital from Venezuela, by phenotypic and genotypic methods.

Methodology
The study was performed with three hundred fifty one (n = 351) carbapenem-resistant or-intermediate clinical isolates recovered during 2016 and 2017 (24month period) in a 192-bed tertiary-care teaching hospital (Dr. Adolfo Pons Hospital) in Maracaibo, Venezuela. The bacterial identification was carried out using conventional methods. A total of one hundred eighty one (n = 181) isolates of K. pneumoniae complex, fifty one (n = 51) P. aeruginosa and one hundred nineteen (n = 119) A. baumannii-calcoaceticus complex were included in the study. Most of the strains were isolates from men (53.08%), among adult patients (20.6%) hospitalized in Intensive Care Units (ICUs) (58.64%). The majority of bacterial species (39.9%) were isolated from VAP (Ventilation Associated Pneumonia), 20.63% from UTI (Urinary Tract Infection) and, 17.42% from bacteremia.
The association with the carbapenemase-producing isolate and the gender and age group of the patients, hospital settings and type of sample was calculated using χ 2 and Fisher's exact tests. p values ≤0.05 were considered to be statistically significant.
To phenotypic detection of carbapenemaseproducing A. baumannii-calcoaceticus complex, the Forty four VIM-type genes were identified in P. aeruginosa isolates (86.27%) and seven in K. pneumoniae complex isolates (3.87%). There was not detection of IMP-type and OXA-type genes ( Table 1).
There was not statistically significant association between the carbapenemase-producing isolate and the gender and age group of the patients, hospital settings and type of sample.

Discussion
The epidemiology of carbapenem resistance in Enterobacterales around the globe has been dominated by the dissemination of three distinct Ambler classes of β-lactamases: NDM, KPC, and OXA-48-like [19].
KPC enzymes are the most prevalent class A carbapenemase worldwide [13] and has been extensively reported in K. pneumoniae and other Enterobacterales [14]. KPC-producing K. pneumoniae is widespread in the United States, but is also endemic in some European countries such as Greece and Italy [14] and were also described in Enterobacterales in the Caribbean, South America, China, and Israel [19].
The first Latin American report of a KPCproducing K. pneumoniae took place in Medellin, Colombia in 2005 and soon afterwards, was also reported all around Latin American countries. In Venezuela, KPC-producing K. pneumoniae and E. cloacae isolates were first described between 2009 and 2010 [4]. We found a clear predominance (77.9%) of K. pneumoniae complex KPC producers, as observed in previous studies describing isolates in Venezuela [6,20].
KPC enzymes have also been identified in other Gram-negative pathogens including P. aeruginosa [14]. The identification of KPC in P. aeruginosa was reported for the first time globally in Colombia in 2007 and KPC-producing A. baumannii-calcoaceticus clinical isolates were described first in Puerto Rico in 2009 [13]; nevertheless, the gene blaKPC has only been identified in K. pneumoniae complex in the present study.
Class B β-lactamases, or metallo-β-lactamases (MBLs), are commonly identified in Enterobacterales and P. aeruginosa. Among the MBLs, NDM, VIM, and IMP enzymes are the most frequently identified worldwide. IMP-producing Gram-negative bacteria are mainly detected in China, Japan, and Australia, mostly in A. baumannii. VIM producers are most often found in Italy and Greece (Enterobacterales) and in Russia (P. aeruginosa) [14].
In Latin America, VIM-2 was identified for first time in 2002 in Chile and Venezuela in isolates of Pseudomonas fluorescens and P. aeruginosa, respectively [7,10], and it has also been reported in P. aeruginosa isolates in Brazil, Argentina, Mexico, Uruguay, Peru and Costa Rica [13]. Overall, in Latin America and the Caribbean, rates of VIM-producing organisms are 2-19% among carbapenem-resistant P. aeruginosa isolates [13], but in the present study, the rate of VIM-producing P. aeruginosa isolates was much more high (86.27%).
The blaVIM gene was also identified in K. pneumoniae complex (3.87%) in this study. In contrast to Europe, few reports of VIM-producing Enterobacterales exist in Latin America and the Caribbean [13]. In Venezuela, VIM-2 was first reported in 2008 among isolates of K. pneumoniae [5] and more recently in Enterobacter spp. [21].
Overall, studies comprising carbapenem-resistant P. aeruginosa isolates in Latin America and the Caribbean have reported rates of IMP production of 1-16% [13]. However, the bla IMP gene was not detected in carbapenemase-producing bacteria in this study.
On the other hand, the MHT is probably the most well-known approach for carbapenemase detection. This assay demonstrates acceptable sensitivity for most carbapenemases, particularly KPC enzymes, but low sensitivity for MBLs [15]. For U.S. collections of Enterobacterales, where KPC producers comprise greater than 95% of carbapenemases [23,24], the sensitivity of the MHT has been reported to be between 93% and 98% [25], like the sensitivity observed in the present study (99%). However, the MHT showed low specificity (45%) in this study for the detection of carbapenemase-producing K. pneumoniae complex. Likewise, MHT showed low sensitivity (50%) and specificity of 71% for detection of P. aeruginosa carbapenemase producers. As isolates producing extended-spectrum β-lactamases (ESBLs) or AmpC cephalosporinases in conjunction with porin mutations often yield false-positive MHT results, the MHT has limited specificity, reported at approximately 91% [10,15]. During the past years, the rates of infections by ESBL-producing isolates have greatly increased. As observed in a previous study in Venezuela, 63.64% of K. pneumoniae isolates were ESBLs producers [6].
The Blue-Carba is a biochemical modified test (BCT) that was validated for the detection of carbapenemase-producing strains directly from bacterial cultures and it detected all Enterobacterales, P. aeruginosa and A. baumannii carbapenemase producers with 100% sensitivity and 100% specificity [23]. In the present study, BCT showed the highest sensitivity and specificity for detection of K. pneumoniae serine carbapenemase producers. To phenotypic detection of carbapenemase-producing A. baumannii-calcoaceticus complex, the BCT showed 63.03% of positive isolates and 31.93% of the isolates were positive in both, BCT and MHT.
Targeted phenotypic carbapenemase assays compare carbapenem activity with and without the presence of inhibitors (e.g., PBA for KPC and EDTA for MBL). Targeted carbapenemase tests offer straightforward, affordable, and accurate options for the detection of KPC, MBL, and OXA-48-like carbapenemase producers. One high stringency evaluation of the assay reported a sensitivity of 97% and specificity of 69% to detect carbapenemase producers. Furthermore, it classified carbapenemases appropriately for 85% of class A, 72% of class B, and 89% of class D carbapenemases [26]. Similar results were obtained in the present investigation; the doubledisk with PBA showed sensitivity of 99% and 38% of specificity for the detection of K. pneumoniae complex carbapenemase producers and sensitivity of 82% and 57% of specificity for the detection of carbapenemaseproducing P. aeruginosa.
OXA-type carbapenemases common to carbapenem-resistant A. baumannii strains are known to be inefficient at hydrolyzing the β-lactam ring of carbapenem antibiotics. The elevated carbapenem MICs observed in carbapenem-resistant A. baumannii strains are largely attributable to additional manifestations of resistance, such as reduced porin expression or upregulation of efflux pumps. The CarbAcineto NP test was designed to overcome some of the impediments associated with detecting carbapenemases produced by A. baumannii, with a sensitivity of 95% and a specificity of 100% for detecting carbapenemases commonly associated with A. baumannii [15].
The spread of CPB remains a significant clinical and public health concern. Consequently, reliable detection of carbapenemase production is an essential first step in combating this problem [15]. The epidemiology of CPB in Venezuela is rapidly evolving, and enhanced surveillance and reporting are needed across the healthcare continuum. Data on regional prevalence rates of carbapenemase-mediated carbapenem resistance are critical for development of coordinated approaches to CPB control [24].
Infections caused by carbapenemase producers are associated with high rates of morbidity and mortality, limited therapeutic options for the treatment, prolonged hospital stays, and overall increased healthcare costs. Therefore, infection prevention remains one of the critical approaches for preventing the spread of such organisms [6,25].

Conclusions
In summary, KPC was the most common carbapenemase detected in K. pneumoniae isolates in Dr. Adolfo Pons´s Hospital in Maracaibo, Venezuela, followed by VIM-producing P. aeruginosa. Finally, considering the increasing identification of carbapenemase-producing bacteria in this hospital, systematic carriage screening at hospital admission, additional surveillance studies, and early detection of such isolates are required to limit their further spread.