1 Introduction

Cholera is described as an enteric disease associated with acute watery diarrhea which results from ingestion of Vibrio cholerae contaminated food or water [1, 2]. Other unhygienic and poor sanitary practices of human are also individuals associated driving factors for spread of cholera infection. The potential pathogen is a Gram negative bacteria that thrives and/or inhabit estuaries and coastal water as free living and colonizer of diverse locale [3, 4]. The case fatality reports of V. cholerae infections (cholera/acute watery diarrhea), variation in pathogenic determinants and changing antibacterial susceptibility profile indicate that the potential pathogen has a tendency to cause disturbing outbreaks if not controlled [1]. This microbial strain as well as other related group members and potential pathogens are said to inhabit estuary/coastal water were the estuary is reported as a black box were pathogens breed resistant genes by some investigators [2, 5,6,7,8,9]. In addition, the environmental water nexus is shown to have been fortified with numerous waste released chemical, disinfecting, cell-free nucleic acids and bactericidal agents [2, 6] which are potential contributing factors to emergence of multiple antibiotic resistance. The distribution of such multiple antibiotic resistant potential pathogens was recently reported in South Africa [2, 5, 7, 10, 11] environment and the possibility for the dissemination of such resistant phenotypes/genotypes further substantiate research interest.

Current area of growing interest has become assessment and report of antibiotic resistance to common and uncommon antibiotics used in the management/control of cholera. However, according to the Clinical Laboratory Standard Institute guidelines [12, 13], important and common antibiotics used for cholera treatment are tetracycline, doxycycline, ampicillin, chloramphenicol, trimethoprime–sulfamethoxazole, azithromycin and ciprofloxacin. Recent reports from various investigators depicts that the potential pathogen is expressing resistance and low susceptibility to these commonly applied antibiotics [14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29], arousing interest in the study. The study reports the occurrence of carbapenemase producing non-serogroup agglutinating V. cholerae (CP-NAG-V.c) in the environment were the carbapenem antibiotics have not been recommended.

2 Materials, methods and experimental details

Various final effluents waste water treatment plants (feWWTPs) and domestic water sources which are located within three municipalities in Eastern Cape Province (S 31° 54.548′, E 026° 50.715ʹ, S 31° 54.548′, E 026° 50.715ʹ, S 31° 54.548′, E 026° 50.715ʹ) were sampled for the study. Water samples from 20 mapped and linked sampling points (including feWWTPs, rivers, receiving water shed, dam, Irrigation-canals and tap water) were retrieved and analyzed since they have similar receiving water shed/water body. Triplicate water samples were collected following standards as recommended [30, 31] into ice cooler boxes and taken for analysis within 8 h at Applied and Environmental Microbiology Research Group (AEMREG) Laboratory at the University of Fort Hare, Alice, South Africa.

2.1 Presumptive bacterial identification

Briefly, yellow/green 18–24 h old bacteria colonies on Thiosulphate-Citrate-Bile-Salts and Sucrose (TCBS/S) agar (Merck KGaA, Darmstadt, Germany) were purified on nutrient agar and stored in fresh aliquot tubes containing 40% glycerol in 2 ml of sterile Luria–Bertani liquid media on sterile 2 ml calibrated and Cryo-skirted-preservation tubes (Starlabs, Milton Keynes, UK)}. Colonial features/characteristic and cultural morphology observed were recorded as Gram negative rods, oxidase positive, motility positive, Voges–Proskauer test positive and d-mannitol catabolism (Table 1). Other in vitro presumptive virulence test including string test, protein hydrolyzing enzyme test, lipid hydrolyzing enzyme test, lecithin hydrolyzase test and cholera red production [2, 32] were also conducted and recorded for the selected Isolates.

Table 1 Cultural, morphological and biochemical characteristics of the bacteria strains
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2.2 Serological detection of isolates

The MAS-AGGL-M11004 Ogawa antisera, MAS-AGGL-M11003 Inaba antisera and MAS-AGGL-M15001 O139 Bengal antisera were commercially obtained from the Mast-Davis diagnostics {Gauteng, South Africa}. An 18–24 h old purified single presumptive colony was smear on clean grease-free slide containing three drops of sterile normal saline. Three drops of each obtained antisera was added and homogenised within one minutes as described in manufacturer instructions. Observation of clumps or agglutination was recorded as positive report depending on the applied antisera (Table 1).

2.3 Antibiotic susceptibility testing of positive/confirmed strains

Identified isolates were grouped based on their sources (feWWTPs, rivers, receiving water shed, dam, and irrigation canal) and subjected to antibiotic sensitivity testing following the guidelines documented by Clinical laboratory Standard Institute guideline [5, 8, 12]. Antibiotic susceptibility testing (AST) was conducted using the disc diffusion method as recommended by Clinical laboratory Standard Institute guideline and other investigators [5, 8, 12]. Antibiotics employed were chloramphenicol, doxycyclin, ampicillin, ceftazidime, cefotaxime, cefazolin, cefuroxime, cefepime, meropenem, doripenem, imipenem and ertapenem. However, based on the nature of the study which focused on carbapenem antibiotic group, only related antibiotics were selectively reported.

2.3.1 Carbapenemase phenotypic and genotypic detection

The study describes the antibiotics used and other methods employed including phenotyping and genotyping of carbapenemase determinants. Test strains with phenotypic resistance profile revealing resistant markers/determinants to the carbapenem antibiotic group; Doripenem (Dor-10 μg), Ertapenem (ETP-10 μg), imipenem (Imi-30 μg), meropenem (Mem-10 μg), and other β-lactam (cephalosporin) antibiotics including ceftriaxone (CRO-30 μg), cefotaxime (CTX-30 μg) and ceftazidime (CAZ-30 μg) were presumptively chosen as carbapenemase producers. The EDTA-Ertapenem synergy investigation was employed to determine presumptive or phenotypic carbapenemase production on previously confirmed nonagglutinating V. cholerae strains [8]. Such strains with positive carbapenemase phenotypic characteristic were confirmed employing the Modified Hodge Test (MHT). Their resistant genotype (New Delhi Metallo-β-lactamase-1 (NDM-1 gene) was further detected using target specific oligonucleotide primer pairs (Sect. 2.4).

2.3.2 EDTA-antibiotic disk diffusion synergy test

This was conducted by first standardizing the test strains on sterile saline suspension broth using 0.5 McFarland standards while the control strains (E. coli ATCC 25922) were treated as test strains in appropriately labeled pre-prepared test tubes. Pre-prepared Mueller–Hinton agar plates (MHAP) were inoculated with standardized suspension and allowed to stand for 10 min. Thereafter, an EDTA-disc which contains 10 µl of pre-prepared 0.5 M-EDTA (approxly; 1.5 mg/disc), Doripenem (Dor-10 μg), Ertapenem (ETP-10 μg) and/or Imipenem (Imi-30 μg) were positioned about 20 mm apart (centre to centre) on pre-prepared agar plates. A negative control test plate which was not fortified with EDTA-disc was also done. Both preparations were incubated at 37 °C for 18–24 h. A positive presumptive carbapenemase producing strains was reported when greater-than 4 mm zone of inhibition was observed on any EDTA fortified disc agar plate were compared to the non-fortified agar plates [5, 8, 9, 12, 33,34,35].

In the same manner, a 6 mm EDTA-disc (containing approxly; 1.5 mg/disc) was applied on Imipenem (IMI-10 µg) disc, placed 10 mm apart and incubated at 37 °C. Positive result is reported with observation of an enlarged clear inhibition zone [5, 8, 9, 12, 33,34,35].

2.3.3 MHT confirmation

The Modified Hodge Test (MHT) was applied as confirmatory test using Meropenem (MEM-10 μg) antibiotic disc on the centre of Mueller–Hinton agar plates while control strain (E. coli ATCC 25922) was treated as test strains. Standardized (0.5 McFarland) test strains were streaked in a straight line lawn across antibiotic disc and allowed to acclimatize for 3–5 min and thereafter incubated at 35 °C ± 2 °C in ambient air overnight. Any record of a clover leaf-type groove at culture intercept was interpreted as positive New Delhi Metallotype-β-lactamase-1 (NDM-1 gene) test.

2.4 Nucleic acid extraction

Nucleic acid (genomic DNA) was extracted from pure isolates using heat-to-boiling technique as previously described by various investigators [8, 36]. The gene-based characterization/detection of serologically positive V. cholerae was done using standard methods and reported elsewhere [8, 9]. Carbapenemase genes were targeted and detected by PCR using target specific primer sequences [37].

2.4.1 Genetic confirmation of blaNDM-1Resistance amongst NAG-V. cholerae

The various presumptive carbapenem enzyme producing strains or β-Metallotype lactamase producers were further confirmed using gene-based PCR detection of blaNDM-1gene. Briefly a 25 µl final volume reaction mixtures was obtained by mixing 15 µl GoTaqPCR master mix reagents (www.promega.com), 0.5 µl of 0.4 µM concentration of each target primer pair {(blaND-MβL-1-F: 5ʹ GGG CAG TCG CTT CCAACG GT 3ʹ and blaND-MβL-1-R: 5ʹ GTA GTG CTC AGT GTC GGC AT 3ʹ)}, 3 µL of extracted template DNA, and 7.0 µl of nuclease-free water was added into sterile 200 µl microfuge tube and loaded into a PCR machine (Bio-Rad T100™ thermal cycler, www.lasec.co.za, SA). The applied cycling condition include: initial denaturation of 94 °C for 2 min; 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and final extension: 72 °C for 10 min and 4 °C post-PCR storage. PCR products were electrophoresed on 1.5% (wt/vol) pre-prepared agarose gel stained with 4 μl/100 mL of 0.5 mg/l ethidium bromide. The observation of an amplicon size of 475 base pair was reported positive for blaNDM-1.

2.5 The study limitations

Following the specification of Clinical laboratory Standard Institute guideline [12] and other standard guidelines for the susceptibility testing of Carbapenem antibiotics, both the disc diffusion test (the Kirby–Bauer method), the micro broth dilution method and/or the agar well diffusion method {as recommended by the European Committee for Antimicrobial Susceptibility Testing (EUCAST) [12, 13, 33, 34]} are needed for the appropriate determination of MIC and susceptibility testing. However, during the study, the micro broth dilution method and/or the agar well diffusion method were not applied for determination of antibiotic susceptibility testing. This was informed by resources available during study. In addition, horizontal transfer of extra-chromosomal DNA (Plasmid) among resistant strains as well as Plasmid profile of such environmental isolates was not also conducted. These areas of the study are currently ongoing in our lab. Furthermore, the whole-genomic and/or partial-genomic sequencing of carbapenem positive strains was not also conducted due to availability of funds. We are currently seeking for research funding (Grant) to cover unattended area of the study and do hope that the reports will be presented/published simultaneously in the future.

2.6 Statistical analysis

The epidemiological statistical tool was applied to determine V. cholerae mean-cell density/count as obtained from experimental water samples, while pearsons correlation was used to determine level of statistical significances in relation to sites (reported elsewhere [8].

3 Results

The results of the study (based on the analysis conducted) are presented as shown below in tables, figures and percentages occurrences. The Table 1 show the cultural, morphological and biochemical characteristics of the bacteria strains recovered during the study. The study showed that among the 943 samples collected, 756 presumptive strains were recovered.

3.1 Serological detection of isolates

The result of various determinate serological analysis conducted on tested isolates were also shown which revealed that among the 756 presumptive isolates recovered, 61 were confirmed as V. cholerae isolates, while 56 (90.2%) were positive to the Ogawa antisera whereas 6 (8.2%) were positive to the inaba antisera. One amongst the strains (1.6%) showed positive agglutination to the Bengal antisera employed during the study. Such observation shows that the bengal strains seldom occur in study environment however this was not the focus of our study.

3.2 Antibiotic susceptibility testing of positive/confirmed strains

The antibiotic susceptibility testing employed three basic strategies which include the usual multidisc antibiotic susceptibility test, EDTA-antibiotic disk diffusion synergy test and MHT Confirmation test. These tests were applied during the study as basic standard and practicable test for carbapenem antibiotic susceptibility testing. Other tests which may have been applied were not conducted due to available funds during study. The Table 2 shows the selected usual multiple disc antibiotic susceptibility testing results while Figs. 1, 2, 3 show the phenotypic resistance and susceptibility profile of isolates with isolates distribution dynamics in the studied water nexus. There was a notable resistance to doripenem as 18% of isolates were resistant to the carbapenem antibiotic member with an intermediate susceptibility report on 23% of isolates. It was also observed that 19.7% of isolates were resistant to imipenem with an intermediate result on 32% of isolates. Furthermore, 34% and 45.9% of isolates had an intermediate susceptibility report to the Ertapenem and Meropenem respectively, while 85.2% of test isolates were resistant to ampicillin. Other detection strategy applied consist a disc of Imipenem, Imipenem-EDTA-disc, Ertapenem and Doripenem with EDTA-disc test, Erterpenem and EDTA-disc test samples as A,B,C,D,E,F are representative photo-microgram of Disk Diffusion Synergy Test. It may be reported that Carbapenemase production and phenotypic detection is shown by a clove expression by strain. It is important to note that only selected antibiotics that address the objective of the study were presented in this report.

Table 2 Shows study prevalence and phenotypic susceptibility profile of isolates
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Fig. 1
figure 1

Presumptive detection of Carbapenemase producing strains using Imipenem and EDTA-disc test. A, B, C, E, F consist a disc of Imipenem, Imipenem-EDTA-disc, while D represents a disc having only Imipenem-EDTA disc

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Fig. 2
figure 2

Presumptive detection of Carbapenemase producing strains using Erterpenem and EDTA-disc test request as samples A, B, C, D are representative photomicrogram of EDTA-Antibiotic Disk Diffusion Synergy Test

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Fig. 3
figure 3

Presumptive detection of Carbapenemase producing strains using Ertapenem and Doripenem with EDTA-disc test. Carbapenemase production and phenotypic detection is shown by a clove expression by strain as shown above

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3.3 Genetic confirmation of blaNDM-1 Resistance amongst NAG-V. cholerae

The results of genetic confirmation/genotyping of blaNDM-1 resistance are shown in Fig. 4 as revealed by the photo-microgram of gel electrophoresis for positive detected somatic antigen nonagglutinating V. cholera strains (SA-NAGVc). The genotypic detection of isolates using target specific New Delhi Metallo-beta-lactamase 1 (ND-MβL-1), which is specific for carbapenemase detection showed representative base pair band size of 475 bp as expected of all isolates producing such resistance genotype which indicates that the tested NAG-V. cholerae strains were producers of carbapenemase and/or carbapenem resistant genotype. The Table 3 and histogram (Fig. 5) shows an analysis conducted to reveal the prevalence of tested strains, distribution dynamics of strains in the study environment and in sampled water nexus. It shows that amongst recovered and SANAG-Vc confirmed strains, 21 isolates were recovered/confirmed from final effluent of waste water treatment plants (feWWTPs), 11 isolates were recovered/confirmed from receiving water shed, 13 isolates were recovered/confirmed from rivers, 6 isolates were recovered/confirmed from dams and 10 isolates were recovered/confirmed from irrigation canal (Table 3, Fig. 5). This is an indication that there is a notable distribution of such strains in addition to resistant phenotype and genotype within the water nexus studied which necessitates the attention of various water wellness regulatory bodies in the studied area water system.

Fig. 4
figure 4

Shows an agarose gel photo of PCR products of representative confirmed New Delhi Metallo-beta-lactamase 1 (ND-MβL-1)amongst NAG-V. cholerae at 475 bp. Lane L: DNA Ladder (100 bp). Lane Pc: The positive control, Lanes 2, 5, 6, 12 are negative while Lanes 1, 3, 4, 7, 8–11, 13 -14 are positive samples

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Table 3 Distribution of Isolates Dynamics in the various water nexus sampled
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Fig. 5
figure 5

Shows distribution dynamics of environmental Isolates in sampled water nexus

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4 Discussion

Diverse determinants of antibiotic resistance have been reported to be present amongst isolates with increasing emergence of newer resistant determinants; one of such is the resistance to carbapenem antibiotic group. The current surge and impact of vibriosis as well as V. cholerae infections in various endemic regions has also added to the threat, as it is revealing emerging resistance virulent determinants. Although carbapenem group of antibiotics are not routinely sourced therapeutic regimen for the control/treatment of V. cholerae related infections, the antibiotic members are currently sourced in some localities that harbors resistant strains of V. cholerae and diverse Vibrio related infections as specified by the CLSI [12]. The current study presents the occurrence of carbapenemase producing NAG Vibrio cholerae (NAG-CPV.c) in aquatic environment before the recommendation of Carbapenem antibiotics. Presumptive Vibrio species recovered during the study were 756 isolates, 61 of these strains were further confirmed as non-agglutinating serogroup (nonO1/nonO139 V. cholerae) using both microbiological methods, serology and molecular biology techniques (Table 1). Amongst the recovered and SANAG-Vc confirmed strains, 21 isolates were recovered/confirmed from final effluent of waste water treatment plants (feWWTPs), 11 isolates were recovered/confirmed from receiving water shed, 13 isolates were recovered/confirmed from rivers, 6 isolates were recovered/confirmed from dams and 10 isolates were recovered/confirmed from irrigation canal (Table 3, Fig. 5). It is important to note that all confirmed/selected isolates were somatic antigen nonagglutinating V. cholerae 1/139 (SANAG-Vc) strains. The feWWTPs isolates were observed to have the highest number of recovered/confirmed isolates implicating the feWWTPs as a potential hub for the distribution of such strains in the water nexus. It is worthy to emphasize that prior to the gene-based confirmation and detection techniques applied, the study also employed both the use of biochemical and serological methods as previously suggested/reported by various related investigators [5, 8, 9, 38,39,40,41]. It was observed that both applied techniques present related positive reports on the strains revealing them as somatic antigen non-agglutinating 1/139 V. cholerae or nonO1/non-O139 V. cholerae strains.

Twenty-five isolates among these confirmed strains were shown to possess carbapenemase phenotype or NDM-1 phenotype (40.89%), (Figs. 1, 2) whereas 24 (39.34%) were observe to produce the MHT positive phenotype (Fig. 3). The application of a gene-based detection strategy for the observed resistant phenotype revealed that 20 (32.79%) were PCR confirmed as NDM-1 resistant gene positive strains (Fig. 4). The observation of such high resistance phenotype/genotype is of potential clinical relevance especially as the carbapenem antibiotic members have not been recommended as a therapeutic regimen for the treatment of V. cholerae infection and other vibriosis within the study region. Furthermore, observing such carbapenem resistant phenotype/genotype in the studied nexus is an indication that there is an existing and ongoing spread as well as sharing of the phenotype/genotype even before the use of the antibiotic group for treatment purpose. Suffice it to say that carbapenemase phenotype/genotype is one amongst the various resistant dynamics that is basically harbored by extra-chromosomal nucleic acids (plasmids) and distributed via horizontal genetic transfer (HGT). Additionally, there is also possibility that any ongoing spread and/or sharing of resistant genes (markers) with relevant resistant phenotype may influence therapeutic management/control of other related bacterial infections. This was the position of some related investigators who opined that because of the increasing heave of carbapenem resistance determinants, there is a significant increase in health concern which have resulted in delayed treatment of related Vibrio infection or other related bacteria cases and a major challenge to treatment [5, 9, 39] of disease cases. Similar reports were published by Shah, his group and other researchers that such observed increase in resistant phenotypes (especially carbapenem which spreads mainly via extra-chromosomal DNA) may result potential increase in the volume and/or incidence of intensive care unit (ICU) cases [9, 42,43,44].

In addition, the PCR detection of such resistant gene (NDM-β-1) among environmental strains of SANAG V. cholerae in water nexus also indicates a potential risk to human, animal, plants, and microbial subjects that applies the water as a source for livelihood. The observed resistant gene also implies that the environmental strains of SANAG- V. cholerae, are emerging with diverse resistant determinants (eg: NDM-1) which also necessitates adroit implementation of appropriate hygienic practice in handling water sources. It has also revealed that there is need for appropriate reporting and documentation of the emerging nature of such resistance determinants especially in water nexus for epidemiological reference in endemic area.

Most recently, the study of diverse investigators [42, 45] did report the occurrence of NDM-1 resistance gene among potential pathogens in water bodies especially amongst Gram-negative strains. NDM-1 resistance genotype and phenotype was also reported among Klebsiella species in patients (hospitalised) in Pakistan and Nepal region with various lineage, plasmid characterization and sequence types [46,47,48,49,50]. However, our study followed a different path where horizontal resistant gene transfer was not described by profiling and detecting resistant plasmid on strains. Although this study structure does not include describing the transferability of resistant genes, the previous reports of related investigators have shown that strains with such resistant gene are potential pathogens of environmental risk. It is therefore, noteworthy for various regulatory organizations and individuals in the study region to employ health related practices and implement novel research based strategies and report any risk related case about users of the water sources.

5 Conclusion

It is evident from the study that carbapenemase producing V. cholerae strains are emerging in the water bodies which necessitates a continuous monitoring and surveillance of the various water bodies using molecular typing technique in major endemic regions. Although the NDM-1 resistant genotypes and phenotypes has been reported to be encoded by plasmid as various related investigators have reported, our study has not confirmed such among the V. cholerae determined. We are hoping to confirm such in our future research as it is an important ongoing research in our lab. However we will not fail to mention that such observation, if true for these V. cholerae strains, would reveal the tendency of strains to disseminate their resistant genotypes among other environmental bacterial strains that co-habit in these water sources and may also contribute to thriving tendency and spread of strains in the estuaries. It is therefore recommended that wastewater regulatory organization as well as nationals should encourage both local and municipal individual wastewater treatment practitioners’ for sustainable management/control and compliance with standard of operations in the near future. Major microbial emerging contaminants of the wastewater final effluent have been described to pose public, clinical and eco-environmental health concerns. Such contaminants/release from non-compliance wastewater works has burdened water re-cycling and reuse as well as irrigation with possible impact on the agricultural production. Important note should be emphasized that any contact and accumulation of microbial contaminants with farm produce (vegetables) may produce and/or increase potential health implications. Consequently, such emerging microbial contaminants (EMC) in effluent release have also been previously reported by related investigators to be associated with high antibiotic resistance in bacteria (ARB) and resistant genes dissemination in the environment, since wastewater serves as a favourable medium/hub for proliferation of both antibiotic resistance and pathogens. Hence, intervention efforts towards controlling the indiscriminate use of antibiotics and unwanted release of effluent into the environment should also be periodically monitored/checked regularly. Furthermore, observing new bacteria implicated antibiotic resistant phenotypes/genotypes such as carbapenem resistance among V. cholerae (CRV.c) strains or New Delhi Metallo β-lactamase producing V. cholerae (NDM-1-V.c) strains and other emerging resistant phenotypes and genotypes observed in this study has revealed the need for another area of research. The need for design/development of potent and better antimicrobial agents (via drug design research) or alternative chemotherapeutics is both imminent and paramount with a view to combat the cholera menace in endemic localities.